Sensor, detection method, detection system, and detection device

ABSTRACT

In an aspect, a sensor includes a combining portion that combines with a second substance having a molecular weight larger than a molecular weight of a first substance. Further, in an aspect, the sensor includes a substrate including a surface on which the combining portion is disposed. The combining portion detects whether or not the first substance is included in an analyte that has come into contact with both an aptamer and the second substance. The aptamer includes a first combining part for the first substance and a second combining part for the second substance and is combined with either of the first substance and the second substance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is national stage application of InternationalApplication No. PCT/JP2013/059658, filed on Mar. 29, 2013, whichdesignates the United States, incorporated herein by reference, andwhich claims the benefit of priority from Japanese Patent ApplicationNo. 2012-080812, filed on Mar. 30, 2012, the entire contents of whichare incorporated herein by reference.

FIELD

The present invention relates to a sensor, a detection method, adetection system, and a detection device.

BACKGROUND

In the past, there is a detection technique of detecting a state changeof a substrate surface. For example, there is a sensor of measuring aproperty or a component of an analyte solution using a surface acousticwave. Further, for example, there is a surface plasmon resonance (SPR)measuring device.

Further, there is a measurement technique using a complex including anaptamer part that is combined with thrombin and inhibits enzyme activityof thrombin and a probe part that is combined with a target molecule. Inthe measurement technique using a complex, for example, when a targetmolecule is combined with a probe part, thrombin is not combined with anaptamer part, and thrombin shows activity. In the measurement techniqueusing a complex, the presence of a target molecule is detected bymeasuring enzyme activity of thrombin.

Further, there is a sensor in which one or more specific nucleic acidligands are attached to a substrate. Further, there is also a sensor inwhich a measuring electrode is coated with an enzyme or the like, and asensor absorbs an analyte solution using a capillary phenomenon.

CITATION LIST Patent Literature

Patent Literature 1: Japanese Patent Application Laid-open No.H05-240762

Patent Literature 2: Japanese Patent Application Laid-open No.2006-184011

Patent Literature 3: Japanese Patent Application Laid-open No.2010-239477

Patent Literature 4: Japanese Patent Application Laid-open No.2005-249491

Patent Literature 5: International Publication Pamphlet No. WO2005/049826

Patent Literature 6: Japanese National Publication of InternationalPatent Application No. 2002-508191

Patent Literature 7: Japanese National Publication of InternationalPatent Application No. 2009-505106

SUMMARY Technical Problem

However, in the detection technique of the related art of detecting thestate change of the substrate surface, there is a problem in thatdetection sensitivity for small molecules having a small molecularweight is bad, and thus it is difficult to detect small molecules.

The technology disclosed herein was made in light of the foregoing, andit is an object of the technology to provide a sensor, a detectionmethod, a detection system, and a detection device, which are capable ofdetecting small molecules.

Solution to Problem

A sensor disclosed herein includes a combining portion that combineswith a second substance having a molecular weight larger than amolecular weight of a first substance. Further, according to one aspect,the sensor includes a substrate including a surface on which thecombining portion is disposed for detecting whether or not the firstsubstance is included in an analyte having come into contact with bothan aptamer and the second substance, the aptamer including a firstcombining part for the first substance and a second combining part forthe second substance and being combined with either of the firstsubstance and the second substance.

Advantageous Effects of Invention

According to an aspect of the detection method according to thedisclosure, an effect capable of detecting small molecule is obtained.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view of a sensor according to an embodiment ofthe present invention.

FIG. 2 is an exploded perspective view of a first cover member and asecond cover member.

FIG. 3 is a perspective view illustrating a state in which a fourthsubstrate of the sensor illustrated in FIG. 1 is taken off.

FIG. 4A is a cross-sectional view taken along line IVa-Iva′ of FIG. 1.

FIG. 4B is a cross-sectional view taken along line IVb-IVb′ of FIG. 1.

FIG. 5 is a perspective view of a detection element used in the sensorillustrated in FIG. 1.

FIG. 6 is a plane view illustrating a state in which a first bondingmember and a second bonding member of the detection element illustratedin FIG. 5 are taken off.

FIG. 7 is a cross-sectional view illustrating a modified example of asensor according to an embodiment of the present invention.

FIG. 8 is a cross-sectional view illustrating another modified exampleof a sensor according to an embodiment of the present invention.

FIG. 9 is a perspective view illustrating an exemplary sensor when acover member is bonded to a substrate.

FIG. 10 is a perspective view illustrating an exemplary sensor wheneither half of a cover member is removed.

FIG. 11A is a cross-sectional view illustrating an exemplary sensor whena cover member is bonded to a substrate.

FIG. 11B is a cross-sectional view illustrating an exemplary sensor whena cover member is bonded to a substrate.

FIG. 12 is a diagram for describing an example of an embodiment of anaptamer according to the disclosure.

FIG. 13 is a diagram for describing a state change of a substratesurface.

FIG. 14 is a diagram for describing another embodiment of a sensor.

FIG. 15 is a diagram for describing a base sequence relation between anATP aptamer and a complementary strand DNA “A.”

FIG. 16 is a diagram illustrating a sensorgram obtained in a BIACORE-Xsystem.

FIG. 17 is a diagram illustrating a sensorgram obtained in a BIACORE-Xsystem.

FIG. 18 is a diagram illustrating sensorgrams obtained in ComparativeExamples 1 to 5 in Table 1.

FIG. 19 is a diagram illustrating sensorgrams obtained in Examples 1 to3 and Comparative Example 6 serving as positive control in Table 1.

FIG. 20 is a diagram illustrating ΔRU in Examples 1 to 9 and ComparativeExamples 6 to 8 serving as positive control in Table 1.

FIG. 21 is a diagram illustrating a third embodiment.

FIG. 22 is a diagram illustrating a fourth embodiment.

FIG. 23 is a diagram for describing an example of an embodiment of anaptamer according to the disclosure.

FIG. 24 is a diagram for describing an example of an embodiment of anaptamer according to the disclosure.

DESCRIPTION OF EMBODIMENTS

Detecting Portion of Sensor, Detection Method, Detection System, andDetection Device According to First Embodiment

Hereinafter, embodiments of a sensor, a detection method, a detectionsystem, and a detection device according to the disclosure will bedescribed in detail with reference to the appended drawings. As will bedescribed below in detail, a sensor, a detection method, a detectionsystem, and a detection device according to the disclosure are capableof detecting a first substance by performing a comparison with a firstsubstance and detecting a state change of a substrate surface caused bya substance having a large molecular weight. As a result, it is possibleto detect a small molecule having a small molecular weight as well.

Hereinafter, a first substance serving as a detection target is alsoreferred to as a “target substance.” Further, when a numerical valuerange is indicated using “to,” it is assumed to include an upper limitvalue and a lower limit value unless otherwise stated. For example, anumerical value range of “300 to 500” indicates that a lower limit is“300 or more,” and an upper limit is “500 or less” unless otherwisestated.

Structure of Sensor

A sensor in which a detecting portion is mounted will be describedbefore the details of a detecting portion of a sensor are described. Thesensor according to the disclosure can be used in a detection techniqueof detecting a state change of a substrate surface. Examples of thesensor according to the disclosure include a measuring cell used formeasurement by a surface plasmon resonance (SPR) device, a surfaceacoustic wave (SAW) sensor, and a quarts crystal microbalance (QCM)crystal sensor. Preferably, the sensor according to the disclosure is anSAW sensor. As the sensor is implemented as the SAW sensor, the sensorcan be simply implemented in small size.

Hereinafter, an exemplary structure of the sensor according to thedisclosure will be described in detail in connection with an example inwhich the sensor according to the disclosure is the SAW sensor. As willbe described below in detail, in an example of an embodiment, a sensor100 serving as the SAW sensor includes a first cover member 1 on which asubstrate 10 is positioned and a second cover member 2 bonded to thefirst cover member 1. Further, in the sensor 100, at least one of thefirst cover member 1 and the second cover member 2 includes an inlet 14through which an analyte flows in, and a flow channel 15 extending fromthe inlet 14 up to at least the surface of the substrate 10 is formedbetween the first cover member 1 and the second cover member 2.

In an example of an embodiment, for example, the sensor 100 includes thefirst cover member 1 on which a substrate 10 is positioned and thesecond cover member 2 bonded to the first cover member 1, and at leastone of the first cover member 1 and the second cover member 2 includesan inlet 14 through which an analyte flows in and a groove portion 15extending from the inlet 14 up to at least the surface of the substrate10. For example, the first cover member 1 includes a concave portionaccommodating at least a part of the substrate 10 on its upper surface,and the second cover member 2 includes the groove portion 15.

In an example of an embodiment, the sensor 100 serving as the SAW sensorfurther includes a first InterDigital Transducer (IDT) electrode thatgenerates an acoustic wave to be propagated to a detecting portion(which will be described in detail later) positioned on the surface ofthe substrate 10. The sensor 100 further includes a second IDT electrodethat is positioned on the surface of the substrate 10 and receives anacoustic wave passing through the detecting portion 13. The sensor 100further includes a first bonding member that is bonded to the uppersurface of the substrate 10 and has a first oscillation spacehermetically-sealed between the first bonding member and the uppersurface of the substrate 10. The sensor 100 further includes a secondbonding member that is bonded to the upper surface of the substrate 10and has a second oscillation space hermetically-sealed between thesecond bonding member and the upper surface of the substrate 10. Here,the first oscillation space is positioned above the first IDT electrode,and the second oscillation space is positioned above the second IDTelectrode.

An exemplary configuration of the sensor 100 serving as the SAW sensorwill be described in detail with reference to the appended drawings. Inthe drawings described below, the same components are denoted by thesame reference numerals. Further, the size of each component, a distancebetween components, and the like are schematically illustrated and maybe different from actual ones. In the sensor 100, any one direction maybe regarded as an upper direction or a lower direction, and hereinafter,for the sake of convenience, an orthogonal coordinate system xyz isdefined, and terms such as an upper surface, a lower surface, and thelike are used under the assumption that a positive side in an zdirection is an upper direction.

The sensor 100 is mainly configured with the first cover member 1, thesecond cover member 2, and a detecting element 3. The first cover member1 includes a first substrate 1 a and a second substrate 1 b stacked onthe first substrate 1 a, and the second cover member 2 includes a thirdsubstrate 2 a stacked on the second substrate 1 b and a fourth substrate2 b stacked on the third substrate 2 a. The detecting element 3 is anSAW element, and is mainly configured with the substrate 10, a first IDTelectrode 11, a second IDT electrode 12, and the detecting portion 13.

The first cover member 1 is attached with the second cover member 2, andthe detecting element 3 is accommodated between the first cover member 1and the second cover member 2 attached to each other. As illustrated incross-sectional views of FIGS. 4A and 4B, the first cover member 1includes a concave portion 5 on its upper surface, and the detectingelement 3 is arranged in the concave portion 5. The second cover member2 includes the inlet 14 serving as an entrance of an analyte solution atits end portion in the longitudinal direction (the x direction), andincludes the groove portion 15 extending from the inlet 14 toward aportion directly above the detecting element 3 as illustrated in FIG. 1.In FIG. 1, in order to indicate the position of the groove portion 15,the groove portion 15 is indicated by a dotted line. An analyte solutionis a solution serving as a detection target as to whether or not a firstsubstance 210 is included therein.

FIG. 2 is an exploded perspective view illustrating the first covermember 1 and the second cover member 2.

First, the first cover member 1 is described.

The first cover member 1 includes the first substrate 1 a and the secondsubstrate 1 b stacked on the first substrate 1 a as described above.

The first substrate 1 a configuring the first cover member 1 has a flatplate shape, and has a thickness of, for example, 0.1 mm to 0.5 mm. Thefirst substrate 1 a has a roughly rectangular plane shape, but its oneend in the longitudinal direction has an arc shape protruding outward.The length of the first substrate 1 a in the x direction is, forexample, 1 cm to 5 cm, and the length in the y direction is, forexample, 1 cm to 3 cm.

The second substrate 1 b is attached to the upper surface of the firstsubstrate 1 a. The second substrate 1 b has a flat plate frame shape inwhich a concave portion forming through hole 4 is formed in a flat plateshaped plate, and has a thickness of, for example, 0.1 mm to 0.5 mm. Anouter shape in a planar view has almost the same as that of the firstsubstrate 1 a, and the length in the x direction and the length in the ydirection are almost the same as those of the first substrate 1 a.

As the second substrate 1 b in which the concave portion forming throughhole 4 is formed is bonded to the first substrate 1 a of the flat plateshape, the concave portion 5 is formed in the first cover member 1. Inother words, the upper surface of the first substrate 1 a positioned onthe inside of the concave portion forming through hole 4 serves as thebottom surface of the concave portion 5, and the inner wall of theconcave portion forming through hole 4 serves as the inner wall of theconcave portion 5.

Further, terminals 6 and interconnections 7 extending from terminals 6to the concave portion forming through hole 4 are formed on the uppersurface of the second substrate 1 b. The terminal 6 is formed on theother end portion of the upper surface of the second substrate 1 b inthe x direction. A portion in which the terminals 6 are formed is aportion that is actually inserted when the sensor 100 is inserted intoan external measuring device (not illustrated), and electricallyconnected to an external measuring device via the terminals 6. Theterminal 6 is electrically connected with the detecting element 3 viathe interconnection 7 or the like. A signal output from the externalmeasuring device is input to the sensor 100 through the terminal 6, anda signal output from the sensor 100 is input to the external measuringdevice through the terminal 6.

Next, the second cover member 2 will be described.

The second cover member 2 includes the third substrate 2 a stacked onthe second substrate 1 b and the fourth substrate 2 b stacked on thethird substrate 2 a as described above.

The second cover member 2 is bonded to the upper surface of the firstcover member 1 configured with the first substrate 1 a and the secondsubstrate 1 b. The second cover member 2 includes the third substrate 2a and the fourth substrate 2 b.

The third substrate 2 a is attached to the upper surface of the secondsubstrate 1 b. The third substrate 2 a has a flat plate shape and has athickness of, for example, 0.1 mm to 0.5 mm. The third substrate 2 a hasa roughly rectangular plane shape, but its one end in the longitudinaldirection has an arc shape protruding outward, similarly to the firstsubstrate 1 a and the second substrate 1 b. The length of the thirdsubstrate 2 a in the x direction is slightly shorter than the length ofthe second substrate 1 b in the x direction so that the terminal 6formed on the second substrate 1 b is exposed, and is, for example, 0.8mm to 4.8 cm. The length in the y direction is, for example, 1 cm to 3cm, similarly to the first substrate 1 a and the second substrate 1 b.

A notch 8 is formed in the third substrate 2 a. The notch 8 is a partformed by cutting out the third substrate 2 a from the apex of one endof the third substrate 2 a having the arc shape toward the other end inthe x direction. The notch 8 serves to form the groove portion 15. Afirst through hole 16 and a second through hole 17 passing through thethird substrate 2 a in the depthwise direction are formed on both sidesof the notch 8 of the third substrate 2 a. When the third substrate 2 ais stacked on the second substrate 1 b, a connection portion between thedetecting element 3 and the interconnection 7 is positioned at theinside of the first through hole 16 and the second through hole 17. Aportion between the first through hole 16 and the notch 8 of the thirdsubstrate 2 a serves as a first partition portion 25 separating thegroove portion 15 (which will be described later) from a space formed bythe first through hole 16. Further, a portion between the second throughhole 17 and the notch 8 of the third substrate 2 a serves as a secondpartition portion 26 separating the groove portion 15 from a spaceformed by the second through hole 17.

The fourth substrate 2 b is attached to the upper surface of the thirdsubstrate 2 a. The fourth substrate 2 b has a flat plate shape and has athickness is, for example, 0.1 mm to 0.5 mm. An outer shape in a planarview is almost the same as that of the third substrate 2 a, and thelength in the x direction and the length in the y direction are almostthe same as those of the third substrate 2 a. As the fourth substrate 2b is bonded with the third substrate 2 a in which the notch 8 is formed,the groove portion 15 is formed below the second cover member 2. Inother words, the lower surface of the fourth substrate 2 b positioned onthe inside of the notch 8 serves as the bottom surface of the grooveportion 15, and the inner wall of the notch 8 serves as the inner wallof the groove portion 15. The groove portion 15 extends from the inlet14 up to at least a portion directly above the detecting portion 13, andhas, for example, a rectangular cross-sectional shape.

A third through hole 18 passing through the fourth substrate 2 b in thedepthwise direction is formed in the fourth substrate 2 b. The thirdthrough hole 18 is positioned above the end portion of the notch 8 whenthe fourth substrate 2 b is stacked on the third substrate 2 a. Thus,the end portion of the groove portion 15 is connected with the thirdthrough hole 18. The third through hole 18 is used to discharge, forexample, air in the groove portion 15 to the outside.

The first substrate 1 a, the second substrate 1 b, the third substrate 2a, and the fourth substrate 2 b are made of, for example, paper,plastic, celluloid, ceramics, or the like. The substrates can be formedof the same material. As all the substrates can be formed of the samematerial, the substances can have the same thermal expansioncoefficient, and thus deformation caused by a thermal expansioncoefficient difference between the substances is prevented. Further, thedetecting portion 13 may be coated with biomaterials, but the detectingportion 13 may be likely to be altered due to external light such asultraviolet light. In this case, it is desirable to use an opaquematerial having a light shielding property as materials of the firstcover member 1 and the second cover member 2. Meanwhile, when thedetecting portion 13 is hardly altered by external light, the secondcover member 2 in which the groove portion 15 is formed may be formed ofnearly a transparent material. In this case, it is possible to view aform of an analyte solution flowing in the flow channel 15.

Next, the detecting element 3 will be described.

FIG. 5 is a perspective view illustrating the detecting element 3, andFIG. 6 is a plane view illustrating the detecting element 3 when a firstbonding member 21 and a second bonding member 22 are taken out.

The detecting element 3 includes the substrate 10, the detecting portion13 arranged on the upper surface of the substrate 10, the first IDTelectrode 11, the second IDT electrode 12, a first extraction electrode19, and a second extraction electrode 20.

For example, the substrate 10 is made of a single crystalline substratehaving piezoelectricity such as a lithium tantalate (LiTaO₃) singlecrystal, a lithium niobate (LiNbO₃) single crystal, or a crystal. Aplane shape and various kinds of dimensions of the substrate 10 may beappropriately set. For example, a thickness of the substrate 10 is 0.3mm to 1 mm.

The first IDT electrode 11 includes a pair of comb electrodes asillustrated in FIG. 6. Each comb electrode includes two bus bars facingeach other and a plurality of electrode fingers extending from each busbar to the other bus bar side. A pair of comb electrodes is arrangedsuch that a plurality of electrode fingers are engaged with one another.The second IDT electrode 12 has the same configuration as the first IDTelectrode 11. The first IDT electrode 11 and the second IDT electrode 12configure a transversal IDT electrode.

The first IDT electrode 11 generates a certain SAW, and the second IDTelectrode 12 receives the SAW generated by the first IDT electrode 11.The first IDT electrode 11 and the second IDT electrode 12 are arrangedin the same straight line form so that the SAW generated by the firstIDT electrode 11 can be received by the second IDT electrode 12. Afrequency characteristic can be designed using the number of electrodefingers of the first IDT electrode 11 and the second IDT electrode 12, adistance between neighboring electrode fingers, a cross width of anelectrode finger, and the like as parameters. As an SAW excited by anIDT electrode, there are waves of various oscillation modes, but in thedetecting element 3, for example, an oscillation mode of a transversalwave called an SH wave is used.

Further, an elastic member for suppressing reflection of an SAW may beinstalled outside the first IDT electrode 11 and the second IDTelectrode 12 in an SAW propagation direction (the y direction). Forexample, a frequency of an SAW can be set within a range from severalmegahertz (MHz) to several gigahertz (GHz). Here, when a frequency of anSAW is set to hundreds of MHz to 2 GHz, it is practical, and downsizingof the detecting element 3 and downsizing of the sensor 100 can beimplemented.

The first IDT electrode 11 is connected with the first extractionelectrode 19. The first extraction electrode 19 extends from the firstIDT electrode 11 to a side opposite to the detecting portion 13, and anend portion 19 e of the first extraction electrode 19 is electricallyconnected with the interconnection 7 formed in the first cover member 1.The second IDT electrode 12 is connected with the second extractionelectrode 20. The second extraction electrode 20 extends from the secondIDT electrode 12 to a side opposite to the detecting portion 13, and anend portion 20 e of the second extraction electrode 20 is electricallyconnected with the interconnection 7.

For example, the first IDT electrode 11, the second IDT electrode 12,the first extraction electrode 19, and the second extraction electrode20 are made of aluminum, an alloy of aluminum and cooper, or the like.The electrodes may have a multi-layer structure. When the electrodeshave a multi-layer structure, for example, a first layer is made oftitanium or chromium, and a second layer is made of aluminum or analuminum alloy.

The first IDT electrode 11 and the second IDT electrode 12 are coveredwith a passivation film (not illustrated). The passivation filmcontributes to anti-oxidation of the first IDT electrode 11 and thesecond IDT electrode 12. For example, the passivation film is formed ofa silicon oxide, an aluminum oxide, a zinc oxide, a titanium oxide, asilicon nitride, or silicon. A thickness of the passivation film isabout a tenth (10 nm to 30 nm) of a thickness of the first IDT electrode11 and the second IDT electrode 12. The passivation film may be formedto cover the entire upper surface of the substrate 10 while exposing theend portion 19 e of the first extraction electrode 19 and the endportion 20 e of the second extraction electrode 20.

The detecting portion 13 is formed between the first IDT electrode 11and the second IDT electrode 12. For example, the detecting portion 13includes a metallic film and an aptamer made of a nucleic acid orpeptide immobilized to a surface of the metallic film. For example, themetallic film has a dual-layer structure including chromium and goldformed on chromium. Examples of the nucleic acid include a deoxyribonucleic acid (DNA), a ribo nucleic acid (RNA), and a peptide nucleicacid (PNA). The details of the detecting portion 13 and the aptamer willbe described later, and a description thereof is omitted.

If the first IDT electrode 11, the second IDT electrode 12 and thedetecting portion 13 arranged in the y direction are assumed to be oneset, two sets are installed on the sensor 100. Thus, as an aptamerimmobilized to one of the detecting portions 13 is made different, it ispossible to perform two types of detections through one sensor. Further,an aptamer may not be immobilized to the other one of the two detectingportions 13 and used as a reference.

The first IDT electrode 11 is covered with the first bonding member 21as illustrated in FIG. 5. The first bonding member 21 is positioned onthe upper surface of the substrate 10, and its inside is hollow. In astate in which the first bonding member 21 is placed on the uppersurface of the substrate 10, the hollow portion of the first bondingmember 21 serves as a first oscillation space 23. The first IDTelectrode 11 is hermetically-sealed in the first oscillation space 23.Thus, the first IDT electrode 11 is isolated from external air and ananalyte solution, and thus the first IDT electrode 11 can be protected.Further, as the first oscillation space 23 is protected, deteriorationsof characteristics of an SAW excited in the first IDT electrode 11 canbe prevented.

Similarly, the second IDT electrode 12 is covered with the secondbonding member 22 as illustrated in FIG. 5. The second bonding member 22is positioned on the upper surface of the substrate 10, similarly to thefirst bonding member 21, and its inside is hollow as illustrated in FIG.4A. In a state in which the second bonding member 22 is placed on theupper surface of the substrate 10, the hollow portion of the secondbonding member 22 serves as a second oscillation space 24. The secondIDT electrode 12 is hermetically-sealed in the second oscillation space24. Thus, the second IDT electrode 12 is isolated from external air andan analyte solution, and thus the second IDT electrode 12 can beprotected. Further, as the second oscillation space 24 is protected,deteriorations of characteristics of an SAW received in the second IDTelectrode 12 can be prevented.

The oscillation space may have a rectangular parallelepiped shape, adome shape when viewed in a cross-sectional view, or an elliptical shapewhen viewed in a planar view, and may have an arbitrary shape accordingto a shape or an arrangement of an IDT electrode or the like.

The first bonding member 21 includes a circular frame body fixed to theupper surface of the substrate 10 to surround the two first IDTelectrodes 11 arranged in the x direction and a cover body fixed to theframe body to block an aperture of the frame body. For example, thisstructure can be formed by forming a resin film using a photosensitiveresin material and patterning the resin film by a photolithographytechnique or the like. The second bonding member 22 can be similarlyformed.

In the sensor 100, the two first IDT electrodes 11 is covered with onefirst bonding member 21, but the two first IDT electrodes 11 may becovered with separate first bonding members 21. Further, the two firstIDT electrodes 11 may be covered with one first bonding member 21, and apartition may be formed between the two first IDT electrodes 11. For thesecond IDT electrode 12, similarly, the two second IDT electrodes 12 maybe covered with separate second bonding members 22, and a partition maybe formed between the two second IDT electrodes 12 using one secondbonding member 22.

A mechanism of detecting a target substance using the detecting element3 using an SAW in the sensor 100 will be described.

In order to detect an analyte solution in the detecting element 3 usingan SAW, a certain voltage is first applied from an external measuringdevice to the first IDT electrode 11 through the interconnection 7, thefirst extraction electrode 19, and the like. In this case, in a regionin which the first IDT electrode 11 is formed, the surface of thesubstrate 10 is excited, and an SAW having a certain frequency isgenerated. A part of the generated SAW is propagated toward thedetecting portion 13, passes through the detecting portion 13, and thenarrives at the second IDT electrode 12. Here, in the detecting portion13, as will be described in detail later, when the analyte solutionincludes the first substance, a change caused by a substance having amolecular weight larger than that of the first substance occurs on thesubstrate surface. As a result, characteristics such as a phase of theSAW passing below the detecting portion 13 change. When the SAW whosecharacteristics have changed as described above arrives at the secondIDT electrode 12, voltage is consequently generated at the second IDTelectrode 12. The voltage is output to the outside through the secondextraction electrode 20, the interconnection 7, and the like, and theexternal measuring device can read the voltage and inspect properties orcomponents of the analyte solution.

In order to guide the analyte solution to the detecting portion 13, thesensor 100 uses a capillary phenomenon. Specifically, since a portion ofthe groove portion 15 formed below the second cover member 2 becomes along thin tube as the second cover member 2 is bonded to the first covermember 1, it is possible to induce a capillary phenomenon to occur inthe long thin tube formed by the groove portion 15 by setting the widthor the diameter of the groove portion 15 to a certain value in view of atype of the analyte solution, materials of the first cover member 1 andthe second cover member 2, and the like. The width (a dimension in the ydirection) of the groove portion 15 is, for example, 0.5 mm to 3 mm, andthe depth (a dimension in the z direction) is, for example, 0.1 mm to0.5 mm. Further, the groove portion 15 includes an extension portion 15e serving as a portion extending beyond the detecting portion 13, andthe third through hole 18 connected to the extension portion 15 e isformed in the second cover member 2. As the analyte solution enters theflow channel 15, air present in the flow channel 15 is discharged to theoutside through the third through hole 18.

As the tube causing the capillary phenomenon is formed in the covermember configured with the first cover member 1 and the second covermember 2, if the analyte solution comes into contact with the inlet 14,the analyte solution is absorbed into the inside of the cover memberusing the groove portion 15 as the flow channel. Thus, according to thesensor 100, since the sensor 100 has a mechanism of absorbing theanalyte solution, it is possible to absorb the analyte solution withoutusing a tool such as a pipette. Further, since the portion in which theinlet 14 is formed is rounded, and the inlet 14 is formed at the apexthereof, the inlet 14 is easily discerned.

Meanwhile, the flow channel 15 of the analyte solution formed by thegroove portion 15 has a depth of about 0.3 mm, the detecting element 3has a thickness of about 0.3 mm, and thus the depth of the flow channel15 is nearly equal to the thickness of the detecting element 3. For thisreason, when the detecting element 3 is placed on the flow channel 15without change, the flow channel 15 is blocked. In this regard, in thesensor 100, as illustrated in FIG. 4, the concave portion 5 is formed inthe first cover member 1 in which the detecting element 3 is mounted,and the detecting element 3 is accommodated in the concave portion 5,and thus the flow channel 15 of the analyte solution is not blocked. Inother words, the flow channel 15 formed by the groove portion 15 can besecured by setting the depth of the concave portion 5 to be nearly equalto the thickness of the detecting element 3 and mounting the detectingelement 3 in the concave portion 5.

FIG. 3 is a perspective view in a state in which the fourth substrate 2b of the second cover member 2 is taken off, but since the flow channel15 of the analyte solution is secured, the analyte solution that hasflown into the flow channel 15 can be smoothly guided to the detectingportion 13 by the capillary phenomenon.

From a point of view of sufficiently securing the flow channel 15 of theanalyte solution, as illustrated in FIG. 4, it is preferable that theheight of the upper surface of the substrate 10 from the bottom surfaceof the concave portion 5 is set to be equal to or smaller than the depthof the concave portion 5. For example, if the height of the uppersurface of the substrate 10 from the bottom surface of the concaveportion 5 is set to be equal to the depth of the concave portion 5, thebottom surface of the flow channel 15 and the detecting portion 13 canhave almost the same height when the inside of the groove portion 15 isviewed from the inlet 14. In the sensor 100, the thickness of thesubstrate 10 is set to be smaller than the depth of the concave portion5, and the height of the first bonding member 21 and the second bondingmember 22 from the bottom surface of the concave portion 5 is set to bealmost equal to the depth of the concave portion 5. If the height of thefirst bonding member 21 and the second bonding member 22 from the bottomsurface of the concave portion 5 is set to be larger than the depth ofthe concave portion 5, it is necessary to process the first partitionportion 25 and the second partition portion 26 of the third substrate 2a to be thinner than other portions, but as the height of the firstbonding member 21 and the second bonding member 22 from the bottomsurface of the concave portion 5 is set to be almost equal to the depthof the concave portion 5, the process is unnecessary, and productionefficiency is good.

The plane shape of the concave portion 5 is similar to, for example, theplane shape of the substrate 10, and the concave portion 5 is slightlylarger than the substrate 10. More specifically, the concave portion 5has the size in which a gap of about 100 μm is formed between the sideof the substrate 10 and the inner wall of the concave portion 5 when thesubstrate 10 is mounted in the concave portion 5.

For example, the detecting element 3 is fixed to the bottom surface ofthe concave portion 5 by a die bond material having epoxy resin,polyimide resin, silicon resin, or the like as a main component. The endportion 19 e of the first extraction electrode 19 is electricallyconnected with the interconnection 7 by a metallic thin line 27 made ofAu or the like. The same also applies in a connection between the endportion 20 e of the second extraction electrode 20 and theinterconnection 7. Further, the first extraction electrode 19 and thesecond extraction electrode 20 may be connected with the interconnection7 by a conductive adhesive material such as Ag-paste as well as themetallic thin line 27.

Gaps are formed in connection portions between the first extractionelectrode 19 and the second extraction electrode 20 and theinterconnection 7. For this reason, the metallic thin line 27 isprevented from being broken when the second cover member 2 is attachedto the first cover member 1. This gap can be simply formed by formingthe first through hole 16 and the second through hole 17 in the thirdsubstrate 2 a. Further, as the first partition portion 25 is presentbetween the first through hole 16 and the groove portion 15, the analytesolution flowing through the groove portion 15 can be prevented fromflowing into the gap formed by the first through hole 16. Thus, it ispossible to prevent a short circuit from occurring by the analytesolution among a plurality of first extraction electrodes 19. Similarly,as the second partition portion 26 is present between the second throughhole 17 and the groove portion 15, the analyte solution flowing throughthe groove portion 15 can be prevented from flowing into the gap formedby the second through hole 17. Thus, it is possible to prevent a shortcircuit from occurring by the analyte solution among a plurality ofsecond extraction electrodes 20.

The first partition portion 25 is positioned on the first bonding member21, and the second partition portion 26 is positioned on the secondbonding member 22. Thus, more technically, the flow channel 15 of theanalyte solution is also defined by the side wall of the first bondingmember 21 at the groove portion side and the side wall of the secondbonding member 22 at the groove portion side as well as the grooveportion 15.

From a point of view for preventing the analyte solution from leaking tothe gaps formed by the first through hole 16 and the second through hole17, it is desirable to cause the first partition portion 25 to come intocontact with the upper surface of the first bonding member 21 and causethe second partition portion 26 to come into contact the upper surfaceof the second bonding member 22, but in the sensor 100, a gap is presentbetween the lower surface of the first partition portion 25 and theupper surface of the first bonding member 21, and between the lowersurface of the second partition portion 26 and the upper surface of thesecond bonding member 22. The gaps are, for example, 10 μm to 60 μm. Asthe gaps are formed, for example, even when the sensor 100 is picked upby fingers and so pressure is applied to the portions, the pressure isabsorbed by the gaps, and thus it is possible to prevent the pressurefrom being applied directly to the first bonding member 21 and thesecond bonding member 22. As a result, the first oscillation space 23and the second oscillation space 24 can be prevented from beingsignificantly deformed. Further, since the analyte solution commonly hasa certain level of viscoelasticity, as the gaps of 10 μm to 60 μm areformed, the analyte solution hardly enters the gaps, and thus theanalyte solution can be prevented from leaking to the gaps formed by thefirst through hole 16 and the second through hole 17.

The width of the first partition portion 25 is set to be larger than thewidth of the first oscillation space 23. In other words, the side wallof the first partition portion 25 is positioned on the frame body of thefirst bonding member 21. Thus, even when the first partition portion 25comes into contact with the first bonding member 21 due to externalpressure, since the first partition portion 25 is supported by the frameportion, the first bonding member 21 can be prevented from beingdeformed. Due to the same reason, the width of the second partitionportion 26 is preferably set to be larger than the width of the secondoscillation space 24.

The first extraction electrode 19, the second extraction electrode 20,the metallic thin line 27, and the interconnection 7 positioned in thegaps formed by the first through hole 16 and the second through hole 17are covered with an insulating member 28. Thus, the electrodes and thelike can be prevented from being corroded. Further, as the insulatingmember 28 is formed, even when the analyte solution enters the gapbetween the first partition portion 25 and the first bonding member 21or the gap between the second partition portion 26 and the secondbonding member 22, the analyte solution is blocked by the insulatingmember 28. Thus, for example, a short circuit between the extractionelectrode caused by leakage of the analyte solution can be prevented.

Thus, according to the sensor 100, as the detecting element 3 isaccommodated in the concave portion 5 of the first cover member 1, theflow channel 15 of the analyte solution from the inlet 14 to thedetecting portion 13 can be secured, and the analyte solution absorbedfrom the inlet by the capillary phenomenon or the like can flow up tothe detecting portion 13. In other words, it is possible to provide thesensor 100 having the absorption mechanism therein while using thedetecting element 3 having the thickness. Further, for example, the flowchannel 15 may include a groove portion formed on the surface of atleast one of the first cover member 1 and the second cover member 2. Inother words, the flow channel 15 may be formed by forming the grooveportion on the surface of at least one of the first cover member 1 andthe second cover member 2.

Modified Example

The above-described structure of the sensor 100 is an example, and thepresent invention is not limited to this example, and an arbitrarysensor 100 may be used.

For example, FIG. 7 is a cross-sectional view illustrating a modifiedexample of the sensor 100. The cross-sectional view corresponds to thecross section illustrated in FIG. 4A. In the modified example, aposition at which the terminal 6 is formed differs. In the aboveembodiment, the terminal 6 is formed at the other end portion of thesecond substrate 1 b in the longitudinal direction, whereas in themodified example, the terminal 6 is formed on the upper surface of thefourth substrate 2 b. The terminal 6 is electrically connected with theinterconnection 7 through a through conductor 29 penetrating through thesecond cover member 2. The through conductor 29 is made of, for example,Ag-paste, plating, or the like. The terminal 6 may be formed on thebottom side of the first cover member 1. Thus, the terminal 6 can beformed at an arbitrary position on the surfaces of the first covermember 1 and the second cover member 2, and it is possible to decide theposition according to a measuring device to be used.

Further, for example, FIG. 8 is a cross-sectional view illustratinganother modified example of the sensor 100. The cross-sectional viewcorresponds to the cross section illustrated in FIG. 4B. In thismodified example, an absorbing material 30 that absorbs the analytesolution at a predetermined speed is disposed at the end of the flowchannel 15 formed by the groove portion 15. As the absorbing material 30is disposed, an excess analyte solution is absorbed, and thusmeasurement can be performed in a state in which the amount of theanalyte solution flowing above the detecting portion 13 is constant andstable. For example, the absorbing material 30 is made of a porousmaterial that can absorb a liquid such as sponge.

The above embodiment has been described in connection with the examplein which the detecting portion 13 includes the metallic film and theaptamer immobilized to the surface of the metallic film, but, forexample, when a substance to be detected by the detecting portion 13reacts with the metallic film, the detecting portion 13 may beconfigured only with the metallic film without using the aptamer.Further, a region between the first IDT electrode 11 and the second IDTelectrode 12 in the surface of the substrate 10 made of a piezoelectricsubstrate may be used as the detecting portion 13 without using themetallic film. In this case, the surface of the substrate 10 comes intodirect contact with the analyte solution to detect physical propertiesof the analyte solution such as viscosity. More specifically, forexample, as the viscosity of the analyte solution on the detectingportion 13 changes, a change in a phase of an SAW is read.

Further, for example, the above embodiment has been described inconnection with the example of the detecting element 3 including an SAWelement, but, for example, the detecting element 3 in which a lightwaveguide or the like is formed to cause an SPR may be used. In thiscase, for example, a change in a refractive index of light is read inthe detecting portion. As another example, the detecting element 3 inwhich an oscillator is formed in a piezoelectric substrate made ofcrystal or the like may be used. In this case, for example, a change inan oscillation frequency of the oscillator is read.

Further, for example, a plurality of types of devices may be mixedlymounted on a single substrate 10 as the detecting element 3. Forexample, an enzyme electrode of an enzyme electrode technique may bedisposed near an SAW element. In this case, it is possible to performmeasurement of an enzymatic technique in addition to an immunizationtechnique using an antibody or an aptamer, and it is possible toincrease the number of items that can be inspected at once.

Further, for example, the above embodiment has been described inconnection with the example in which the first cover member 1 isconfigured with the first substrate la and the second substrate 1 b, andthe second cover member 2 is configured with the third substrate 2 a andthe fourth substrate 2 b, but the present invention is not limited tothis example, and a cover member in which substrates are integrated, forexample, the first cover member 1 in which the first substrate 1 a isintegrated with the second substrate 1 b may be used.

Further, for example, the above embodiment has been described inconnection with the example in which one detecting element 3 isinstalled, but a plurality of detecting elements 3 may be installed. Inthis case, the concave portion 5 may be formed for each detectingelement 3, and the long concave portion 5 capable of accommodating allthe detecting elements 3 may be formed.

Further, for example, the groove portion 15 may be formed in either orboth of the first cover member 1 and the second cover member 2. In otherwords, the flow channel 15 may be formed by forming grooves in both thefirst cover member 1 and the second cover member 2, and the flow channel15 may be formed by forming a groove in either of the first cover member1 and the second cover member 2.

Further, for example, FIGS. 9 to 11 are diagrams illustrating aconfiguration in which a cover member 45 is bonded directly to thesubstrate 10. The above embodiment has been described in connection withthe example in which the substrate 10 is disposed on the first covermember 1, and the first cover member 1 is bonded with the second covermember 2, but the present invention is not limited to this example. Forexample, the flow channel 15 may be formed such that the cover member isbonded directly to the substrate 10. The details will be describedbelow.

FIGS. 9 to 11 will be described in connection with an example in whichthe flow channel 15 is formed by forming a groove in the cover member 45bonded to a substrate 10A. The present invention is not limited to thisconfiguration, and, for example, the flow channel 15 may be formed byforming grooves in both of the cover member 45 disposed on the uppersurface of the substrate 10A and the substrate 10A, and the flow channel15 may be formed by forming a groove in the substrate 10A.

FIG. 9 is a perspective view illustrating an exemplary sensor when acover member is bonded to the substrate. In the example illustrated inthe example illustrated in FIG. 9, the sensor 100A includes a substrate10A and a cover member 45. The cover member 45 includes an inlet 14Aserving as an inlet of the analyte solution and a third through hole 18Aserving as an air hole or an outlet of the analyte solution. Further, inthe example illustrated in FIG. 9, the inlet 14A is formed in the uppersurface of the cover member 45, but the present invention is not limitedto this example. For example, the inlet 14A may be formed in the side ofthe cover member 45, similarly to the sensor 100. Furthermore, in theexample illustrated in FIG. 9, the cover member 45 includes pads 44. Thepads 44 correspond to the end portion 19 e of the first extractionelectrode 19 and the end portion 20 e of the second extraction electrode20 of the sensor 100.

FIG. 10 is a perspective view illustrating an exemplary sensor wheneither half of a cover member is removed. FIG. 10 is a perspective viewof the sensor 100A when either half of the cover member 45 is removed.As illustrated in FIG. 10, a space 40 serving as an analyte flow channelof the analyte solution is formed in the cover member 45. The inlet 14Ais connected with the space 40. In other words, the analyte solutioninput from the inlet 14A flows into the space 40. The space 40 in thesensor 100A corresponds to the flow channel 15 in the sensor 100.

FIGS. 11A and 11B are cross-sectional views illustrating an exemplarysensor when a cover member is bonded to a substrate. FIG. 11A is across-sectional view taken along line IVa-IVa of FIG. 9, and FIG. 11B isa cross-sectional view taken along line IVb-IVb of FIG. 9.

As illustrated in FIGS. 11A and 11B, a first IDT electrode 11, a secondIDT electrode 12, a short circuit electrode 42 a, a short circuitelectrode 42 b, and the like are formed on the upper surface of thesubstrate 10A. Further, the first IDT electrode 11, the second IDTelectrode 12, the short circuit electrode 42 a, the short circuitelectrode 42 b, and the like are covered with a passivation film 41. Thepassivation film 41 contributes to anti-oxidation of the electrodes andthe interconnections. The passivation film 41 is made of, for example, asilicon oxide, an aluminum oxide, a zinc oxide, a titanium oxide, asilicon nitride, or silicon. For example, the passivation film 41 ismade of a silicon dioxide (SiO2).

The passivation film 41 is formed to cover the entire upper surface ofthe substrate 10A while exposing the pads 44. As the first IDT electrode11 and the second IDT electrode 12 are covered with the passivation film41, corrosion of the IDT electrodes can be prevented.

A thickness of the passivation film 41 is, for example, 100 nm to 10 μm.The passivation film 41 need not necessarily be formed to cover theentire upper surface of the substrate 10A, and for example, thepassivation film 41 may be formed to cover only a portion around thecenter of the upper surface of the substrate 10A while exposing a regionalong an outer periphery of the upper surface of the substrate 10Aincluding the pads 44. Further, FIGS. 11A and 11B illustrate the examplein which the passivation film 41 is used, but the present invention isnot limited to this example, and the passivation film 41 may not beused.

The short circuit electrode 42 a and the short circuit electrode 42 bserve to cause a portion of the upper surface of the substrate 10Aserving as an SAW propagation channel to be electrically shortcircuited. As the short circuit electrode 42 a and the short circuitelectrode 42 b are disposed, SAW loss can be reduced according to a typeof SAW. Further, particularly, when a leaky wave is used as an SAW, aloss suppression effect by the short circuit electrode 42 a and theshort circuit electrode 42 b is considered to be increased.

For example, the short circuit electrode 42 a or the short circuitelectrode 42 b has a rectangular shape extending along the SAWpropagation channel from the first IDT electrode 11 to the second IDTelectrode 12. For example, the width of the short circuit electrode 42 aor the short circuit electrode 42 b in a direction (the x direction)orthogonal to an SAW propagation direction is equal to a cross width ofthe electrode fingers of the first IDT electrode 11. Further, the endportion of the short circuit electrode 42 a or the short circuitelectrode 42 b at the first IDT electrode side in a direction (the ydirection) parallel to the SAW propagation direction is positioned at adistance of a half wavelength of an SAW from the center of the electrodefingers positioned at the end portion of the first IDT electrode 11.Similarly, the end portion of the short circuit electrode 42 a or theshort circuit electrode 42 b at the second IDT electrode 12 in the ydirection is positioned at a distance of a half wavelength of an SAWfrom the center of the electrode fingers positioned at the end portionof the second IDT electrode 12.

Here, frequency characteristics can be designed using the number ofelectrode fingers of the first IDT electrode 11 and the second IDTelectrode 12, a distance between neighboring electrode fingers, a crosswidth of the electrode finger, and the like as parameters. Examples ofthe SAW excited by the IDT electrode include a Rayleigh wave, a lovewave, and a leaky wave. Further, an elastic member for anti-reflectionof an SAW may be disposed in a region outside the first IDT electrode 11in the SAW propagation direction. For example, the frequency of an SAWcan be set within a range from several megahertz (MHz) to severalgigahertz (GHz). Here, when a frequency of an SAW is set to hundreds ofMHz to 2 GHz, it is practical, and downsizing of the substrate 10A anddownsizing of the sensor 100A can be implemented.

The short circuit electrode 42 a or the short circuit electrode 42 b maybe in an electrically floating state, or a pad 44 for ground potentialmay be disposed, and the short circuit electrode 42 a or the shortcircuit electrode 42 b may be connected to the pad 44 and have groundpotential. When the short circuit electrode 42 a or the short circuitelectrode 42 b has ground potential, propagation of a direct wave byelectromagnetic coupling between the first IDT electrode 11 and thesecond IDT electrode 12 can be suppressed.

For example, the short circuit electrode 42 a or the short circuitelectrode 42 b may be made of aluminum, an alloy of aluminum and copper,or the like. The electrodes may have a multi-layer structure. When theelectrodes have a multi-layer structure, for example, a first layer ismade of titanium or chromium, and a second layer is made of aluminum oran aluminum alloy.

Plate-like bodies 43 have concave portions for forming the firstoscillation space 23 and the second oscillation space 24, and as theplate-like bodies 43 are bonded to the substrate 10A, the firstoscillation space 23 and the second oscillation space 24 are formed. Forexample, the plate-like bodies 43 are formed using a photosensitiveresist. The plate-like bodies 43 correspond to the first bonding member21 and the second bonding member 22 in the sensor 100. In the exampleillustrated in FIGS. 11A and 11B, a penetrating portion penetratingthrough the plate-like bodies 43 in the depthwise direction is formedbetween the concave portions of the plate-like bodies 43 for forming thefirst oscillation space 23 and the second oscillation space 24. Thepenetrating portion is formed to form a metallic film on an SAWpropagation channel. In other words, when the plate-like body 43 isbonded to the substrate 10A, if viewed in a planar view, at least a partof the propagation channel of an SAW propagating from the first IDTelectrode 11 to the second IDT electrode 12 is exposed from thepenetrating portion, and the detecting portion 13 is formed in theexposed portion.

Further, for example, an arbitrary process may be performed on thedetecting portion 13. For example, when one of the two detectingportions 13 is used as a reference, a process of preventing a substancedetected by the detecting portion 13 from being attached to the metallicfilm used as the reference may be performed. A concrete example in whichthe detecting portion 13 is combined with a nucleic acid such as DNAwill be described. In this case, since a nucleic acid such as DNA isnegatively changed, by negatively charging the metallic film of thedetecting portion 13 used as a reference through an arbitrary technique,it is possible to prevent a nucleic acid such as DNA from beingerroneously attached to the reference. Similarly, since a nucleic acidsuch as DNA tends to be attached to gold, a metallic film made of metalother than gold may be used as the metallic film of the detectingportion 13 used as the reference. Further, for example, when a signalsubstance is a nucleic acid, a nucleic acid of a random arrangement maybe immobilized to the reference to be same as at the detection side. Asa result, as the surface state of the reference becomes the same as thesurface state of the detection side, it is possible to cancel, forexample, a small difference in viscosity considered to be caused by adifference between surfaces, and it is possible to regard onlycombination at the detection side as a difference between the referenceand the detection side.

The sensor according to the above embodiment and the sensors accordingto various kinds of modified examples are effective in detecting a smallmolecule, and can be used for the general purpose for maintaining beautyor youth such as a fatigue or anti-aging marker as well as for thepurpose for use in an existing medical system such as a cancer marker.Here, an SAW chip serving as a high-sensitivity transducer is embeddedas a disposable sensor, and a signal substance (or an aptamer itself)dissociated from an aptamer is combined with/disassociated from asubstrate surface in a capillary flow channel on the SAW chip, and thusit is possible to implement a sensor that is high in sensitivity to asmall molecule, disposable, light in weight, compact, and small in size.As a result, it is possible to implement a small simple sensor.

For example, a variable structure type aptamer mass change detectingportion is disposed on an SAW propagation channel, and thus propagationof an SAW interacts with a signal substance or an aptamer. As a result,for example, it is possible to directly detect a mass change in which anamount of a target detection object is increased, it is easy to performconversion for quantification, and it is possible to amplify a signaland perform detection with a high degree of accuracy. Further, a signalsubstance or an aptamer is larger in mass than a small molecule to bedetected, and a detection result can be amplified.

Further, for example, a SAW propagation channel serving as a portionreacting with a biological substance and an IDT electrode serving as aportion performing conversion into an electric signal can be finelymanufactured on a single substrate. As a result, a very small sensor canbe implemented, mass production is possible in a wafer process or thelike, and a disposable sensor chip can be simply implemented.

Further, for example, an SAW detecting circuit has a circuitconfiguration similar to that employed in communication devices such asmany wireless terminals or tablet terminals, and thus the detectingcircuit of the sensor can be simply connected to an electronic devicesuch as a wireless terminal or a tablet terminal.

Detecting Portion of Sensor According to First Embodiment

In an embodiment, the sensor 100 according to the disclosure includesthe substrate 10. In an embodiment, the sensor 100 according to thedisclosure further includes a combining portion 240, and the combiningportion 240 is positioned on the surface of the substrate 10, can becombined with a second substance 220 having a molecular weight largerthan a molecular weight of the first substance 210, and can detectwhether or not the first substance 210 is included in an analyteincluding the second substance 220 and an aptamer 230 that can becombined with the first substance 210 and the second substance 220. Thesecond substance 220 is also referred to as a “signal substance.”

For example, the sensor 100 includes the combining portion 240 that iscombined with the second substance 220 having a molecular weight largerthan that of the first substance 210. Further, for example, in thesensor 100, the combining portion 240 detecting whether or not the firstsubstance 210 is included in the analyte that has come into contact withboth the aptamer 230 that includes a first combining part 231 for thefirst substance 210 and a second combining part 232 for the secondsubstance 220 and is combined with either of the first substance 210 andthe second substance 220 and the second substance 220 is disposed on thesurface of the substrate 10.

Here, the first substance 210 is an arbitrary substance. For example,the first substance 210 is a low molecular (small molecular) organiccompound such as protein, an enzyme, a cell, a cell tissue,microorganism, a virus, a bacterium, toxin, nucleic acid, a saccharide,a lipid, metabolite, or an adenosine TriPhosphate (ATP). Further, forexample, the first substance 210 is an arbitrary substance serving as amarker indicating a body condition such as stress, fatigue, and variouskinds of diseases, induced pluripotent stem cell (iPS), or the like.

The second substance 220 is an arbitrary substance having a molecularweight larger than that of the first substance 210. For example, thesecond substance 220 is an enzyme, protein, a nucleic acid, or the like.More preferably, the second substance 220 is a nucleic acid. Using anucleic acid, it is possible to simply control bond strength between thesecond combining part 232 and the second substance 220 by changing abase number forming a complementary strand.

Here, the molecular weight of the first substance 210 and the molecularweight of the second substance 220 are supplementarily described. Thesecond substance 220 is preferably a substance having a molecular weightlarger than the first substance 210. For example, the molecular weightof the second substance 220 is 10,000 or more. For example, themolecular weight of the first substance 210 is 500 or less, and morepreferably 200 to 500. Here, the present invention is not limited tothis example, the molecular weights of the first substance 210 and thesecond substance 220 may have an arbitrary value.

The aptamer refers to a substance that is high in affinity for aspecific substance and specifically combined with a specific substance.The following description will proceed with an example of using anucleic acid aptamer serving as the aptamer 230 formed of a nucleicacid, but the present invention is not limited to this example, apeptide aptamer may be used, and an arbitrary aptamer 230 may be used.Further, when a nucleic acid aptamer is used, various kinds ofmodifications may be performed on a nucleic acid forming a nucleic acidaptamer. Further, an exemplary base sequence of the aptamer 230 in whichthe first substance 210 is ATP is indicated by a sequence number 1.

FIG. 12 is a diagram for describing an exemplary aptamer according to anembodiment of the disclosure. In the example illustrated in FIG. 12, forconvenience of description, in addition to the aptamer 230, the firstsubstance 210 and the second substance 220 are illustrated. Further, forconvenience of description, FIG. 12 will be described in connection withan example in which the aptamer 230 combined with the second substance220 is mixed with the analyte solution, and the aptamer 230 and thesecond substance 220 come into contact with the analyte solution. Here,the present invention is not limited to this example, and by causing theaptamer 230 and the second substance 220 to be attached to the flowchannel 15 of the sensor in advance, the analyte solution coming intocontact with the aptamer 230 and the second substance 220 may come intocontact with the detecting portion 13. At this time, the aptamer 230combined with the second substance 220 in advance may be combined withthe side of the flow channel 15 so that the aptamer 230 is not separatedfrom the side of the flow channel 15. Further, by causing the aptamer230 and the second substance 220 to be attached to or combined with theside of the flow channel 15 of the sensor at the inlet side rather thanthe detecting portion 13, the aptamer 230 and the second substance 220may be caused to come into contact with the analyte solution with a highdegree of accuracy before arriving at the detecting portion 13.

In other words, for example, since the aptamer 230 and the secondsubstance 220 are attached to the groove portion 15, the combiningportion 240 may detect the first substance 210 from the analyte that hascome into contact with the aptamer 230 and the second substance 220being attached to the groove portion 15. Further, for example, since theaptamer 230 is immobilized to the groove portion 15 and combined withthe second substance 220, the combining portion 240 may detect the firstsubstance 210 from the analyte that has come into contact with theaptamer 230. Here, for example, the aptamer 230 being combined with thesecond substance 220 is chemically combined with a surface substance ofthe groove portion 15.

Here, at least one of the aptamer 230 and the analyte may be positioned,away from the combining portion 240. Further, at least one of theaptamer 230 and the analyte may be positioned in the groove portion 15.Furthermore, at least one of the aptamer 230 and the analyte may bepositioned in the detecting portion.

Further, when the aptamer 230 and the second substance 220 are attachedto the flow channel 15 in advance, it is important to employ a dry formand cause the aptamer 230 and the second substance 220 to be attached tothe flow channel 15 in a separable form so that at least the secondsubstance 220 is separated from the flow channel 15 by contact with theanalyte solution and comes into contact with the detecting portion 13together with the analyte solution.

(1) of FIG. 12 illustrates an example in which the first substance 210is not included in the analyte solution, and (2) of FIG. 12 illustratesan example in which the first substance 210 is included in the analytesolution.

As illustrated in FIG. 12, the aptamer 230 according to the disclosureincludes the first combining part 231 for the first substance 210 andthe second combining part 232 for the second substance 220, and iscombined with either of the first substance 210 and the second substance220.

As illustrated in FIG. 12, the aptamer 230 includes the first combiningpart 231 that is combined with the first substance 210 and the secondcombining part 232 that is combined with the second substance 220. Asillustrated in (1) of FIG. 12, the aptamer 230 is designed such that thesecond combining part 232 is combined with the second substance 220 whenthe first substance 210 is not present in the analyte solution. On theother hand, as illustrated in (2) of FIG. 12, the aptamer 230 isdesigned such that the first combining part 231 is combined with thefirst substance 210, and the second substance 220 combined with thesecond combining part 232 is disassociated from the aptamer 230 when thefirst substance 210 is present in the analyte solution. In other words,when the first substance 210 is not present in the analyte solution, theaptamer 230 and the second substance 220 form a complex. Meanwhile, whenthe first substance 210 is present in the analyte solution, the aptamer230 is disassociated from the second substance 220, and the aptamer 230is combined with the first substance 210 to form a complex. In otherwords, the aptamer 230 is designed to be combined with the firstsubstance 210 in preference to the second substance 220.

Here, a mechanism in which when the first substance 210 is present inthe analyte solution, the first combining part 231 is combined with thefirst substance 210, and the second substance 220 combined with thesecond combining part 232 is disassociated from the aptamer 230 issupplementarily described. For example, the second substance 220 beingcombined with the second combining part 232 is disassociated from thefirst combining part 231 due to influence on a steric structure of theaptamer 230 caused by a combination of the first substance 210 and thefirst combining part 231, influence of a steric barrier by the firstsubstance 210 combined with the first combining part 231, or the like.Here, a mechanism in which the second substance 220 is disassociatedfrom the aptamer 230 is not limited to this example, and an arbitrarymechanism may be used.

Here, a design technique of designing the aptamer 230 having themechanism in which when the first substance 210 is present in theanalyte solution, the first combining part 231 is combined with thefirst substance 210, and the second substance 220 combined with thesecond combining part 232 is disassociated from the aptamer 230 issupplementarily described. The following description will proceed withan example of using single-strand DNA having a molecular weight of about15,000 as the second substance 220 for convenience of description, butthe present invention is not limited to this example, and RNA, PNA, oran arbitrary substance may be used as the second substance 220. Anexemplary technique of deciding the base sequence the first combiningpart 231, an exemplary technique of deciding the base sequence of thesecond combining part 232, and an exemplary technique of deciding thebase sequence of the aptamer 230 will be described below in thedescribed order.

An exemplary technique of deciding the base sequence of the firstcombining part 231 is now described. For example, the base sequence ofthe first combining part 231 may be decided by an in vitro selectiontechnique, a systematic evolution of ligands by exponential enrichment(SELEX) technique, or the like. In further detail, the base sequence ofthe first combining part 231 or the second combining part 232 is decidedsuch that the aptamer 230 that is specifically combined with the firstsubstance 210 is acquired by the in vitro selection technique or theSELEX technique, and the base sequence of the acquired aptamer 230 isdecoded. Here, the present invention is not limited to this example. Forexample, when the first substance 210 is a nucleic acid, a base sequencecomplementary to a part or whole of the nucleic acid serving as thefirst substance 210 may be used instead of the in vitro selectiontechnique, the SELEX technique, or the like.

An exemplary technique of deciding the base sequence of the secondcombining part 232 is now described. The base sequence of the secondcombining part 232 may be decided in a manner similar to the firstcombining part 231. Further, when a nucleic acid is used as the secondsubstance 220, a base sequence complementary to a part or whole of anucleic acid serving as the second substance 220 may be used as the basesequence of the first combining part 231 instead of the in vitroselection technique, the SELEX technique, or the like. Here, as thesecond substance 220, a substance having a molecular weight larger thanthat of the first substance 210 is used, and more preferably, asubstance having a molecular weight of 10,000 or more is used. Thus,when the base sequence complementary to the whole of the nucleic acidserving as the second substance 220 is used, combination strength is toolarge, and it is not disassociated, and thus it is desirable to use thebase sequence complementary to a part of the nucleic acid as the secondcombining part 232.

An exemplary technique of deciding the base sequence of the aptamer 230is now described. The base sequence of the aptamer 230 is decided basedon the base sequence of the first combining part 231 and the basesequence of the second combining part 232. For example, the basesequence of the aptamer 230 may be decided so that the base sequence ofthe first combining part 231 is terminally combined with the basesequence of the second combining part 232. Here, the present inventionis not limited to this example, and the base sequence in which the basesequence of the second combining part 232 is inserted into the basesequence of the first combining part 231 may be used, and the basesequence in which the base sequence of the first combining part 231 isinserted into the base sequence of the second combining part 232 may beused.

Further, the second combining part 232 may be formed by a part of thebase sequence of the first combining part 231. In this case, the secondsubstance 220 is a nucleic acid having a base sequence complementary tothe second combining part 232 serving as a part of the first combiningpart 231. Further, the second combining part 232 may be formed by a partof a base sequence of the first combining part 231 at the 3 terminalside or the 5 terminal side and a base sequence independent of thesecond combining part 232.

Further, an arbitrary base sequence may be used as a base sequence otherthan the part of the base sequence of the second substance 220complementary to the second combining part 232. Preferably, the basesequence other than the part complementary to the second combining part232 may be a base sequence that does not have a sequence complementaryto a part or whole of the first combining part 231 or the secondcombining part 232. For example, a base sequence in which one arbitrarybase continues up to the terminal may be used.

Here, the base number of the second combining part 232 issupplementarily described. As the base number of the second combiningpart 232 increases, combination strength between the second combiningpart 232 and the second substance 220 increases. As a result, as thebase number of the second combining part 232 increases, the secondcombining part 232 is more likely to be combined with the secondsubstance 220, and when the first substance 210 is combined with thefirst combining part 231, the second substance 220 is unlikely to bedisassociated from the second combining part 232. Similarly, as the basenumber of the second combining part 232 decreases, the combinationstrength between the second combining part 232 and the second substance220 decreases. As a result, as the base number of the second combiningpart 232 decreases, the second combining part 232 is unlikely to becombined with the second substance 220, and when the first substance 210is combined with the first combining part 231, the second substance 220is likely to be disassociated from the second combining part 232. Forthis reason, the base number of the second combining part 232 has anappropriate range, and is preferably “20” or less, and more preferably“9 to 11.”

The analyte solution may be an arbitrary solution including a liquid ora solid serving as a detection target, and the analyte solution comesinto contact with the aptamer 230 and the second substance 220 as theaptamer 230 and the second substance 220 are added to and mixed in theanalyte solution in advance, or the analyte solution passes through theflow channel 15 of the sensor 100 and comes into contact with theaptamer 230 and the second substance 220 attached or immobilized to theflow channel 15. Further, for the ratio of the aptamer 230 and thesecond substance 220 coming into contact with the analyte solution,preferably, the mole ratio of the aptamer 230 is equal to or larger thanthe mole ratio of the second substance 220, and more preferably, themole ratio of the aptamer 230 is equal to the mole ratio of the secondsubstance 220.

Further, the combining portion 240 for the second substance 220 fordetecting whether or not the first substance 210 is included in theanalyte solution is disposed on the surface of the substrate 10. Thedetecting portion 13 may be the entire substrate surface or a part of asubstrate surface. For example, the detecting portion 13 includes themetallic film and the combining portion 240 immobilized to the metallicfilm. Here, the present invention is not limited to this example, andthe detecting portion 13 may not include the metallic film. When thedetecting portion 13 includes the metallic film, any metal may be usedto form the metallic film. For example, preferably, Au (gold), Ti, Cu,or the like is used, and more preferably, gold is used.

Here, the combining portion 240 of the substrate surface is described.An arbitrary substance that is specifically combined with the secondsubstance 220 is used as the combining portion 240 of the substratesurface. For example, an aptamer, protein, or an antibody which isspecifically combined with the second substance 220 is used as thecombining portion 240 of the substrate surface. For example, thecombining portion 240 of the substrate surface is decided in a mannersimilar to the second combining part 232. Here, an arbitrary substancethat is specifically combined with the second substance 220 is notlimited to the example of using an aptamer, protein, or an antibody. Forexample, when a material forming a substrate surface is combined withthe second substance 220, a separate aptamer, protein, or an antibodymay not be used.

An immobilizing technique of immobilizing the combining portion 240 tothe substrate surface is now described. An arbitrary technique may beused as the immobilizing technique. For example, immobilization may beperformed using strong affinity between streptavidin and biotin. In thiscase, for example, streptavidin is immobilized to the detecting portion13 in advance. In further detail, streptavidin is immobilized onto thesurface on which a self-assembled monolayer (SAM) made of alkylthiol orthe like is formed in advance to cover the surface (Au or the like) ofthe detecting portion 13 as much as possible when immobilized. Further,biotin is immobilized to an end portion of a substance used as thecombining portion 240 of the substrate surface in advance, and asolution of a substance used as the combining portion 240 of thesubstrate surface is prepared. Thereafter, the solution including thesubstance used as the combining portion 240 of the substrate surface iscaused to come into contact with the detecting portion 13, and thus thecombining portion 240 is immobilized to the detecting portion 13.Thereafter, in order to remove a substance that is not immobilized tothe detecting portion 13 and remains in the detecting portion 13, thedetecting portion 13 may be cleaned using an arbitrary solvent. Forexample, NaOH is used as a solvent used for cleaning. Here, a solventused for cleaning is not limited to NaOH, and an arbitrary solvent maybe used.

Here, a relation among the first substance 210, the aptamer 230, and thecombining portion 240 is supplementarily described. The first substance210, the aptamer 230, and the combining portion 240 have a magnituderelation related to a free energy change. Specifically, there is amagnitude relation in which a first free energy change calculated from adissociation constant of the first substance 210 and the aptamer 230 issmaller than a second free energy change associated with a combinationof the aptamer 230 and the second substance 220. Further, there is amagnitude relation in which a third free energy change associated with acombination of the second substance 220 and the combining portion 240 islarger than the second free energy change.

In other words, the first free energy change calculated from thedissociation constant of the first substance 210 and the aptamer 230 issmaller than the second free energy change associated with a combinationof the aptamer 230 and the second substance 220, and the third freeenergy change associated with a combination of the second substance 220and the combining portion 240 is larger than the second free energychange.

The free energy indicates a free energy change of Gibbs and has anegative value. It is because when a spontaneous reaction occurs, a freeenergy has a negative value. As the free energy change of Gibbsincreases in a negative direction, a reaction is more likely to proceed.In other words, for example, the “magnitude relation in which the firstfree energy change is smaller than the second free energy change”indicates a magnitude relation in which both the first free energychange and the second free energy change have a negative value, and anabsolute value of the first free energy change is larger than anabsolute value of the second free energy change.

Here, the description will proceed with an example in which the aptamer230 and the combining portion 240 have a base sequence complementary toa part of the base sequence of the second substance 220. Further, thedescription will proceed with an example in which the combining portion240 has a base sequence complementary to a part of the base sequence ofthe second substance 220. Furthermore, the description will proceed withan example in which the second free energy is a free energy changeassociated with a combination of the complementary parts of the basesequence of the aptamer 230 and the base sequence of the secondsubstance 220. Moreover, the description will proceed with an example inwhich the third free energy is a free energy change associated with acombination of the complementary parts of the base sequence of thesecond substance 220 and the base sequence of the combining portion 240.In this case, the base type and the base number of the complementarybase sequence between the aptamer 230 and the second substance 220 andthe base type and the base number of the complementary base sequencebetween the combining portion 240 and the second substance 220 havevalues satisfying the magnitude relation among the first free energychange, the second free energy change, and the third free energy change.

In other words, the description will proceed with an example in whicheach of the aptamer 230 and the combining portion 240 have the basesequence. Further, the description will proceed with an example in whichthe base sequence of the aptamer 230 has a part complementary of a firstpart of the base sequence of the second substance 220, and the basesequence of the combining portion 240 has a part complementary to asecond part of the base sequence of the second substance 220.Furthermore, the description will proceed with an example in which thesecond free energy change is a free energy change associated with acombination of the complementary parts of the first part of the basesequence of the second substance 220 and the base sequence of theaptamer 230. The description will proceed with an example in which thethird free energy change is a free energy change associated with acombination of the complementary parts of the second part of the basesequence of the second substance 220 and the base sequence of thecombining portion 240. In this case, the base type and the base numberof the complementary base sequence between the aptamer 230 and thesecond substance 220 and the base type and the base number of thecomplementary base sequence between the combining portion 240 and thesecond substance 220 have values satisfying the magnitude relation amongthe first free energy change, the second free energy change, and thethird free energy change.

Further, the aptamer 230 may be combined with or attached to the SAWpropagation channel or the flow channel 15 in advance, and may be meltedand included in the analyte solution before the analyte solution flowsinto the flow channel 15 of the sensor 100. For example, at least one ofthe aptamer 230 and the analyte is positioned away from the combiningportion 240 or the detecting portion 13. Further, for example, at leastone of the aptamer 230 and the analyte is positioned in the flow channel15. Furthermore, for example, at least one of the aptamer 230 and theanalyte is positioned in the combining portion 240 or the detectingportion 13.

Further, a change in a propagation constant of an SAW is limited to achange in a surface of a substrate. As a result, for example, a processof removing a non-reacted substance that has not reacted with a targeteven when the non-reacted substance remains on the substrate is notparticularly necessary. Through an operation of simply causing theanalyte solution to flow into the capillary flow channel, a signalsubstance or an aptamer associated with a combination with a target iscombined with/disassociated from (a part functioning as) a receptor(such as a combining portion) immobilized to the substrate surface, andthus it is possible to selectively detect influence by combining anddisassociating.

Detection Method According to First Embodiment

In an embodiment, a detection technique according to the disclosureincludes a contact process of causing an analyte to come into contactwith the surface of the substrate 10 of the sensor having the combiningportion 240 for the second substance 220, and the analyte includes thefirst combining part 231 for the first substance 210 and the secondcombining part 232 for the second substance 220 having a molecularweight larger than that of the first substance 210, and comes intocontact with both the aptamer 230 that is combined with either of thefirst substance 210 and the second substance 220 and the secondsubstance 220.

Further, when the aptamer 230, the second substance 220, and the analyteare added to and mixed in the analyte solution in advance, mixing may beperformed using an arbitrary technique. For example, the aptamer 230,the second substance 220, and the analyte may be mixed and prepared, theaptamer 230 in which the second substance 220 is combined with thesecond combining part 232 in advance and the analyte may be mixed andprepared, or an arbitrary technique may be used.

Here, when the aptamer 230, the second substance 220, and the analyteare added to and mixed in the analyte solution in advance, preferably,the aptamer 230 in which the second substance 220 is combined with thesecond combining part 232 in advance and the analyte solution may bemixed and prepared. Using the aptamer 230 in which the second substance220 is combined with the second combining part 232 in advance, althoughthe first substance 210 is not present, the second substance 220 is notcombined with the aptamer 230, and thus it is possible to reduce aphenomenon that the second substance 220 is combined with the combiningportion 240 and improve a detection accuracy.

An arbitrary technique may be used as a technique of combining thesecond substance 220 with the second combining part 232 in advance. Thedescription will continue with an example in which the second substance220 is a nucleic acid, and the second combining part 232 is a nucleicacid complementary to the second combining part 232. In this case, sincethe second substances 220 do not form a double strand, and the secondcombining parts 232 of the aptamer 230 do not form a double strand, thesecond substance 220 may be combined with the second combining part 232in advance by mixing and stirring at a room temperature. Further, forexample, by heat denaturation under a reaction condition in a polymerasechain reaction (PCR) technique, a double strand DNA is converted into asingle strand with a high degree of accuracy, and a primer is annealedon a single strand DNA with a high degree of accuracy. For this reason,the second substance 220 may be combined with the second combining part232 in advance using a temperature condition in the PCR technique. Infurther detail, the second substance 220 may be combined with the secondcombining part 232 in advance such that the second substance 220 ismixed with the aptamer 230, and then the mixture of the second substance220 and the aptamer 230 is heated to a temperature at which a doublestrand DNA is converted into a single strand by heat denaturation andthereafter cooled.

Further, a technique of causing the analyte solution to come intocontact with the substrate surface may be an arbitrary technique. Forexample, when the sensor 100 serving as the SAW sensor is used, theanalyte solution may be caused to come into contact with the substratesurface by guiding the analyte solution from the inlet 14 to thedetecting portion 13 via the groove portion 15 as described above.Further, when the sensor is a measuring cell of an SPR device or a QCMcrystal sensor, the analyte solution may be manually caused to come intocontact with a substrate surface of a biocell, or the analyte solutionmay be caused to come into contact with a flow cell of the SPR device orthe QCM measuring device by injecting the analyte solution into the flowcell.

Further, the detection technique according to the disclosure includes adetecting process of detecting the first substance 210 from the analyteby detecting the state change of the surface of the substrate 10 withwhich the analyte has come into contact. FIG. 13 is a diagram fordescribing a state change of a substrate surface. FIG. 13 will bedescribed in connection with an example in which, for convenience ofdescription, an analyte solution for detection is prepared by mixing theaptamer 230 being combined with the second substance 220 with theanalyte solution. (1) of FIG. 13 illustrates an example in which thefirst substance 210 is not included in the analyte solution, and (2) ofFIG. 13 illustrates an example in which the first substance 210 isincluded in the analyte solution. In FIG. 13, for example, a metalliclayer serving as the detecting portion 13 positioned on the uppersurface of the substrate 10 is not illustrated.

As illustrated in (1) of FIG. 13, when the first substance 210 is notpresent in the analyte solution, the aptamer 230 is combined with thesecond substance 220 through the second combining part 232, and thus thecombining portion 240 immobilized to the substrate surface is notcombined with the second substance 220. On the other hand, asillustrated in (2) of FIG. 13, when the first substance 210 is presentin the analyte solution, the first combining part 231 is combined withthe first substance 210, and the second substance 220 being combinedwith the first combining part 231 is disassociated from the aptamer 230.Then, the disassociated second substance 220 is combined with thecombining portion 240 immobilized to the substrate surface.

Here, the state change of the substrate surface refers to a mass change,a dielectric constant change, a viscoelasticity change, a propagationcharacteristics change, a resonance frequency change, and the like whichare caused as the combining portion 240 immobilized to the substratesurface is combined with the second substance 220. For example, whenmeasuring is performed using the SPR device, if the combining portion240 immobilized to the substrate surface is combined with the secondsubstance 220, a mass or a dielectric constant of the substrate surfacechanges, and due to this change, an SPR angle change occurs. In thiscase, the state change of the substrate surface is a mass change or adielectric constant change caused by a combination of the combiningportion 240 and the second substance 220, and the state change of thesubstrate surface is detected by detecting the SPR angle change.Further, when the SAW sensor is used, a propagation characteristicschange is caused by a mass change or a viscoelasticity change of thesubstrate surface. In this case, the state change of the substratesurface is a mass change or a viscoelasticity change caused by acombination of the combining portion 240 and the second substance 220,and the state change of the substrate surface is detected by detectingthe propagation characteristics change. Further, when the QCM measuringdevice is used, a resonance frequency change is caused by a mass changeof the substrate surface. In this case, the state change of thesubstrate surface is the mass change caused by a combination of thecombining portion 240 and the second substance 220, and the state changeof the substrate surface is detected by detecting the resonancefrequency change.

The change of the substrate surface is caused by a combination of thecombining portion 240 immobilized to the substrate surface and thesecond substance 220, and the combining portion 240 immobilized to thesubstrate surface is combined with the second substance 220 when thefirst substance 210 is included in the analyte solution. Further, thesecond substance 220 has a molecular weight larger than a molecularweight of the first substance 210. As a result, compared to thetechnique of detecting the change of the substrate surface caused by acombination of the first substance 210 and the substrate surface, thetechnique of detecting the change of the substrate surface caused by acombination of the second substance 220 and the substrate surface islarger in a mass change, a dielectric constant change, and aviscoelasticity change in the substrate surface and thus can improve thedetection sensitivity. Accordingly, it is possible to detect a smallmolecule that is hardly measured by the technique of the related art ofimmobilizing a small molecule to a substrate surface and detecting asmall molecule.

Detection System and Detection Device According to First Embodiment

In an embodiment, the detection system according to the disclosureincludes a sensor including a combining portion 240 for a secondsubstance 220 having a molecular weight larger than that of a firstsubstance 210 and a substrate 10 including the combining portion 240disposed on its surface.

Here, the sensor used in the first embodiment of the detection systemand the detection device is the same as the above-described sensor, anda description thereof is omitted.

Next, in an embodiment, the detection system according to the disclosurefurther includes a detection device. The detection device includes adetection control unit that detects whether or not the first substance210 is included in the analyte by detecting the state change of thesurface of the substrate 10 when the analyte coming into contact withthe aptamer 230 that includes the first combining part 231 for the firstsubstance 210 and the second combining part 232 for the second substance220 and combines with either of the first substance 210 and the secondsubstance 220 and the second substance 220 comes into contact with thesurface of the substrate 10 of the sensor including the combiningportion 240 for the second substance 220 having a molecular weightlarger than that of the first substance 210 and the substrate 10 havingthe combining portion 240 disposed on its surface.

The detection device is a device that performs an arbitrary detectionprocess using the sensor. Examples of the detection device include theSPR device, a control device of the SAW sensor, and the QCM measuringdevice. Preferably, the detection device is the control device of theSAW sensor. The SPR device, the control device of the SAW sensor, andthe QCM measuring device serving as the detection device according tothe disclosure may be an arbitrary device capable of performingmeasurement using the sensor, or a known device may be used withoutchange or may be appropriately modified and then used.

Further, not a target substance, but a signal substance combined withthe substrate surface or an aptamer disassociated from the substratesurface is detected by the sensor. For this reason, the detection devicemay perform a conversion process of converting a detection resultobtained based on the signal substance or the like into a detectionresult of the target substance. For example, when a molecular weight ofthe target substance and a molecular weight of the signal substance arealready known, if a result indicating that “the signal substance is “x”gram (or mol),” the result may be converted into a result indicting thatthe target substance is “y” gram (or mol).”

Detecting Portion of Sensor, Detection Method, Detection System, andDetection Device According to Second Embodiment

The first embodiment has been described in connection with the examplein which the change of the substrate surface caused by the secondsubstance 220 having a molecular weight larger than that of the firstsubstance 210 instead of the first substance 210 is detected. Here, thesensor according to the disclosure is not limited to this example.

FIG. 14 is a diagram for describing another embodiment of a sensor. Inother words, for example, as illustrated in (1) of FIG. 14, an aptamer300 having a molecular weight larger than that of a first substance 210is combined with a combining portion 310 of a substrate surface inadvance. Then, as illustrated in (2) of FIG. 14, when the firstsubstance 210 is included in the analyte solution, the first substance210 may be combined with the aptamer 300, the aptamer 300 may bedisassociated from the combining portion 310 of the substrate surface,and the state change of the substrate surface caused by disassociationof the aptamer 300 may be detected.

The following description will proceed focusing on points different fromthe detecting portion of the sensor, the detection method, the detectionsystem, and the detection device according to the first embodiment.

A sensor according to a second embodiment includes a substrate 10. Thesensor further includes the combining portion 310 that is positioned ona surface of the substrate 10 and is being combined with the aptamer 300that can be combined with the first substance 210. Here, the combiningportion 310 can detect whether or not the first substance 210 isincluded.

For example, the sensor includes the combining portion 310 for theaptamer 300 including a combining part that is combined with the firstsubstance 210. Further, for example, the sensor includes the substrate10 in which the combining portion 310 that detects whether or not thefirst substance 210 is included in the analyte is disposed on itssurface, and the aptamer 300 being combined with either of the firstsubstance 210 and a combining portion 240 is combined with the combiningportion 310.

In other words, similarly to the aptamer 230, the aptamer 300 includestwo combining parts, and one of the combining parts is combined with thefirst substance 210, and the other is combined with the combiningportion 310 of the substrate surface. In other words, the aptamer 300includes a first combining part 231 that is combined with the firstsubstance 210 and a combining part 321 that is combined with thecombining portion 310. For example, when the combining portion 310 isformed of a nucleic acid, a nucleic acid having a base sequencecomplementary to the combining portion 310 is used as the combining part321 of the aptamer 300 that is combined with the combining portion 310.

Further, a detection method according to the second embodiment includesa contact process of causing an analyte to come into contact with thesurface of the substrate 10 of the sensor including the combiningportion 310, the substrate 10 in which the combining portion 310 isdisposed on its surface, and the aptamer 300 that is combined with thecombining portion 310, includes the combining part that is combined withthe first substance 210, and is combined with either of the firstsubstance 210 and the combining portion 310. In other words, thedetection method according to the second embodiment includes a contactprocess of causing the analyte solution to come into contact with thesubstrate surface of the sensor 100 in which the aptamer 300 thatincludes the combining part that is combined with the first substance210 and is combined with either of the combining portion 310 of thesubstrate surface of the sensor and the first substance 210 is combinedwith the combining portion 310. The detection method according to thesecond embodiment further includes a detecting process of detectingwhether or not the first substance 210 is included in the analyte bydetecting the state change of the surface of the substrate 10 with whichthe analyte comes into contact by the contact process.

Further, the detection system according to the second embodiment includea sensor including the combining portion 310, the substrate 10 in whichthe combining portion 310 is disposed on its surface, and the aptamer300 that is combined with the combining portion 310, includes thecombining portion 310 that is combined with the first substance 210, andis combined with either of the first substance 210 and the combiningportion 310. In other words, the detection system according to thesecond embodiment includes the sensor 100 in which the aptamer 300 thatincludes the first combining part 231 that is combined with the firstsubstance 210 and is combined with either of the combining portion 310of the substrate surface of the sensor 100 and the first substance 210is combined with the combining portion 310. The detection systemaccording to the second embodiment further includes a detection devicethat detects whether or not the first substance 210 is included in theanalyte by detecting the state change of the surface of the substrate 10when the analyte comes into contact with the surface of the substrate 10of the sensor.

The detection device according to the second embodiment includes adetection control unit that detects whether or not the first substance210 is included in the analyte by detecting the state change of thesurface of the substrate 10 when the analyte comes into contact with thesurface of the substrate 10 of the sensor including the combiningportion 310, the substrate 10 in which the combining portion 310 isdisposed on its surface, and the aptamer 300 that is combined with thecombining portion 310, includes the combining portion 310 that iscombined with the first substance 210, and is combined with either ofthe first substance 210 and the combining portion 310. In other words,the detection device according to the second embodiment includes adetection control unit that detects whether or not the first substance210 is included in the analyte solution by detecting the state change ofthe substrate surface when the analyte solution comes into contact withthe substrate surface of the sensor 100 in which the aptamer 300 thatincludes the combining part that is combined with the first substance210 and is combined with either of the combining portion 310 of thesubstrate surface of the sensor 100 and the first substance 210 iscombined with the combining portion 310.

Detecting Portion of Sensor, Detection Method, Detection System, andDetection Device According to Third Embodiment

The above second embodiment has been described in connection with theexample in which the aptamer 300 having a molecular weight larger thanthat of the first substance 210 is combined with the combining portion310 of the substrate surface in advance, but the present invention isnot limited to this example.

FIG. 21 is a diagram illustrating a third embodiment.

(1) of FIG. 21 illustrates a state in which there are a plurality ofaptamers 300. (2) of FIG. 21 illustrates a state in which an analytesolution including a plurality of first substances 210 comes intocontact with the aptamer 300. (3) of FIG. 21 illustrates a state inwhich the aptamer 300 coming into contact with an analyte solutionincluding the first substance 210 comes into contact with a plurality ofcombining portions 310 positioned on a substrate 10.

In other words, for example, the combining portion 310 is not combinedwith the aptamer 300 in advance, and after the analyte solution comesinto contact with the aptamer 300, the analyte solution comes intocontact with the combining portion 310. Here, when the first substance210 is included in the analyte solution, the first substance 210 iscombined with the aptamer 300. As a result, among the aptamers 300included in the analyte solution, the aptamer 300 that has not beencombined with the first substance 210 is combined with the combiningportion 310.

In the example illustrated in (1) of FIG. 21, there are four aptamers300. Here, as illustrated in (2) of FIG. 21, when the first substance210 is present in the analyte solution, the aptamer 300 is combined withthe first substance 210. For example, in the example illustrated in (2)of FIG. 21, three of the four aptamers 300 are combined with the firstsubstance 210. Thereafter, as illustrated in (3) of FIG. 21, in thecombining portion 310 of the surface of the substrate 10, the aptamer300 not being combined with the first substance 210 is combined with thecombining portion 310, and the aptamer 300 being combined with the firstsubstance 210 is not combined with the combining portion 310. In otherwords, for example, when the first substance 210 is not present in theanalyte solution, each of the four aptamers 300 illustrated in (1) ofFIG. 21 is combined with the combining portion 310, whereas, asillustrated in (2) and (3) of FIG. 21, when the first substance 210 ispresent in the analyte solution, the number of aptamers 300 to becombined with the combining portion 310 is reduced by the number ofaptamers 300 that have been combined with the first substance 210.

Thus, when the first substance 210 is included in the analyte solution,the number of aptamers 300 to be combined with the combining portion 310is smaller than when the first substance 210 is not included in theanalyte solution. Similarly, as the number of first substances 210included in the analyte solution increases, the number of aptamers 300to be combined with the combining portion 310 decreases. In the thirdembodiment, it is possible to detect the state change of the surface ofthe substrate 10 caused by a combination of the aptamer 300 and thecombining portion 310.

The third embodiment will be described in further detail. A sensoraccording to the third embodiment includes a substrate 10 and acombining portion 310 that is disposed on the substrate 10, and iscombined with an aptamer 300 including a combining part that is combinedwith a first substance 210.

Here, in the third embodiment, it is detected whether or not the firstsubstance 210 is included in the analyte solution by causing the analytesolution that comes into contact with the aptamer 300 to come intocontact with the substrate 10 in which the combining portion 310 isdisposed on its surface and detecting the state change of the surface ofthe substrate 10 coming into contact with the analyte solution.Specifically, based on the fact that when the first substance 210 isincluded in the analyte solution, a small number of aptamers 300 arecombined with the combining portion 310 compared to when the firstsubstance 210 is not included in the analyte solution, it is detectedwhether or not the first substance 210 is included in the analytesolution. Further, similarly, based on the fact that when the firstsubstance 210 is included in the analyte solution, as the number offirst substances 210 included in the analyte solution increases, thenumber of aptamers 300 to be combined with the combining portion 310decreases, an amount of the first substances 210 included in the analytesolution is measured.

Here, for example, an amount of the aptamers 300 is more preferablyequal to or larger than an upper limit of the number of moles in aconcentration range of an analyte to be measured with respect to aconcentration of an analyte, that is, the number of moles of the firstsubstance 210 (to be combined with the aptamer 300). Further, thecombining portion 310 is preferably immobilized to the substrate 10 inas high density as possible at which the aptamer 300 is combined withthe combining portion 310 without being saturated when the firstsubstance 210 is not combined with the aptamer 300. It is to make itpossible to appropriately obtain a change in a state of the substratesurface according to a concentration of an analyte in a concentrationrange of an analyte to be measured.

As described above, in the third embodiment, even when a concentrationof the first substance 210 is low, it is possible to obtain a signal forthe concentration at an excellent SN.

Detecting Portion of Sensor, Detection Method, Detection System, andDetection Device According to Fourth Embodiment.

Further, for example, a relation between the aptamer and the secondsubstance with respect to the combining portion of the substrate 10 maybe changed.

FIG. 22 is a diagram illustrating a fourth embodiment.

(1) of FIG. 22 illustrates a state in which in the analyte solution,there are an aptamer 430 and a second substance 420, but there is nofirst substance 410. (2) of FIG. 22 illustrates a state in which in theanalyte solution of (1) of FIG. 22, there is the first substance 410 inaddition to the aptamer 430 and the second substance 420. In otherwords, in the fourth embodiment, as illustrated in FIG. 22, thesubstrate 10 includes a combining portion 440 that is complementarilycombined with the aptamer 430. The aptamer 430 includes a firstcombining part 431 that is combined with the second substance 420 and asecond combining part 432 that is combined with the combining portion440.

Here, the description will proceed with an example in which a firstsubstance 410 is smaller in a molecular weight than the aptamer 430, andhas a combining ability of combining with the second substance 420stronger than a combining ability of combining with the aptamer 430. Inthis case, when the analyte solution including the aptamer 430 and thefirst substance 410 comes into contact with the surface of the substrate10, the second substance 420 is disassociated from the aptamer 430, andcombined with the first substance 410. Then, the aptamer 430 from whichthe second substance 420 has been disassociated is combined with thecombining portion 440, and the state of the surface of the substrate 10is changed.

For example, as illustrated in (1) of FIG. 22, when the first substance210 is not present in the analyte solution, the aptamer 430 isdesignated such that the first combining part 431 of the aptamer 430 iscombined with the second substance 420. On the other hand, asillustrated in (2) of FIG. 22, when the first substance 210 is presentin the analyte solution, the aptamer 430 is designed such that thesecond substance 420 being combined with the aptamer 430 is combinedwith the first substance 410, and disassociated from the aptamer 430.Thereafter, the second combining part 432 of the aptamer 430 from whichthe second substance 420 has been disassociated is combined with thecombining portion 440 disposed on the surface of the substrate 10.

In other words, in the fourth embodiment, when the first substance 410is included in the analyte solution, the combining portion 440 iscombined with the aptamer 430, and it is detected that the state of thesurface of the substrate 10 is changed. In other words, the firstsubstance 410 included in the analyte solution is detected by detectingthe change in the state of the surface of the substrate 10 caused by acombination of the combining portion 440 and the aptamer 430.

Aptamer

The above embodiments have been described in connection with the examplein which, for example, as illustrated in FIG. 12, the first combiningpart 231 for the first substance 210 and the second combining part 232for the second substance 220 are formed at different positions in theaptamer 230. Here, the present invention is not limited to this example,and the first combining part 231 may overlap the second combining part232 in whole or part. In other words, in the aptamer 230, at least apart of the first combining part 231 and at least a part of the secondcombining part 232 may be the same part. The aptamer 230 ispreferentially combined with either of the first substance 210 and thecombining portion 240.

FIGS. 23 and 24 are diagrams for describing exemplary aptamers accordingto an embodiment of the disclosure. As illustrated in FIGS. 23 and 24,for example, the first combining part 231 may overlap the secondcombining part 232 in whole or part, or both the first substance 210 andthe second substance 220 may be combined with the same part. In theexample illustrated in FIG. 23, in the aptamer 230, the second substance220 is combined with one surface of a solid line part, and the firstsubstance 210 is combined with the other surface of the solid line part.In other words, the solid line part of FIG. 23 includes the firstcombining part and the second combining part. Further, in the exampleillustrated in FIG. 24, the first combining part 231 partially overlapsthe second combining part 232. In other words, as illustrated in FIG.24, the first combining part 231 is indicated by a dotted line, thesecond combining part 232 is indicated by a solid line, and the firstcombining part 231 partially overlaps the second combining part 232.Thus, the aptamers 230 of FIGS. 23 and 24 have the same function as thatof the aptamer 230 of FIG. 12.

EXAMPLES

Hereinafter, examples will be described in further detail in connectionwith an example in which in the sensor, the detection method, thedetection system, and the detection device according to the disclosure,an ATP is used as the first substance, and the SPR device is used as ameasuring device. Here, the sensor, the detection method, the detectionsystem, and the detection device according to the disclosure are notlimited to the following examples.

In the following, a sensor chip SA (GE Healthcare) is used as a sensor.The sensor chip SA is a chip used for SPR measurement by a BIACORE-Xsystem. In the sensor chip SA, streptavidin is immobilized onto asubstrate in advance through carboxymethyl dextran. The followingdescription will proceed with an example of the aptamer 230 using an ATPas the first substance 210. Hereinafter, the aptamer 230 using an ATP asthe first substance 210 is referred to as an “ATP aptamer.”

Examples 1 to 9

In Examples 1 to 9, as will be described below in detail, an ATP aptamercomplementary strand DNA mixed liquid was prepared. Further, a biotinDNA solution was prepared. Thereafter, immobilization of biotin DNA ontothe sensor chip SA and checking of the immobilization were performed,and SPR measuring was performed.

Preparation of ATP Aptamer Complementary Strand DNA Mixed Liquid

A mixed liquid of an ATP aptamer having a base sequence described as asequence number 1 in the sequence listing and one of DNAs “A” to “C”having base sequences described as sequence numbers 3 to 5 in thesequence listing was prepared. The ATP aptamer and the DNAs “A” to “C”were obtained by custom synthesis (Gene Design Inc.). Hereinafter, theDNAs “A” to “C” are also referred to as complementary strand DNAs “A” to“C,” respectively.

Sequence number 1: 5′-ACCTGGGGGAGTATTGCGGAGGAAGGT-3′ Sequence number 3:5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACCTTCCTCC-3′ Sequence number 4:5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACCTTCCTCCGC-3′ Sequence number 5:5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACCTTCCTCCGCAA-3′

Specifically, first, an ATP aptamer 80 μl of 10 μM was mixed with acomplementary strand DNA 40 μl to 10 μM. Then, annealing of the ATPaptamer and the complementary strand DNA was performed such that themixed liquid of the ATP aptamer and the complementary strand DNA[x] washeated for one minute at a temperature of 95°, heated for one minutes ata temperature of 75°, and then placed for 30 minutes at a roomtemperature.

Here, as illustrated in FIG. 15, the base sequence of the ATP aptamerand the base sequence of the complementary strand DNA “A” arecomplementary to each other from the 3 terminal side to the 10 basesequence. FIG. 15 is a diagram for describing a base sequence relationbetween the ATP aptamer and the complementary strand DNA “A.” Similarly,the base sequence of the ATP aptamer and the base sequence of thecomplementary strand DNA “B” are complementary to each other from the 3terminal side to the 12 base sequence. Further, the base sequence of theATP aptamer and the base sequence of the complementary strand DNA “C”are complementary to each other from the 3 terminal side to the 14 basesequence. As described above, the complementary strand DNAs “A” to “C”differ in combination force with the ATP aptamer. Specifically, thecombination force increase in the order of the complementary strandsDNAs “C,” “B,” and “A.”

Preparation of Biotin DNA Solution

A biotin DNA solution 200 μl was prepared by mixing a biotin DNA 1 μl of10 mM with a HBS-N buffer (BIACORE) 199 μl. The biotin DNA has a basesequence described as the sequence number 2 in the sequence listing, andhas biotin added to the 5 terminal side of the base sequence. A finalconcentration of the biotin DNA was 5 μM. The biotin DNA was obtained bycustom synthesis (Gene Design Inc.).

Sequence number 2: 5′-GGAGGAAGGT-3′

Immobilization of Biotin DNA to Sensor Chip and Checking ofImmobilization

The biotin DNA was immobilized to the sensor chip SA using strongaffinity between streptavidin and biotin. Further, SPR measuring wasperformed using the BIACORE-X (GE Healthcare Japan Corporation), and theimmobilization of the biotin DNA was checked. The following conditionwas used when the measuring was performed using the BIACORE-X system.

Running buffer: HBS-N buffer (BIACORE)

Velocity: 5 μl/min

Temperature: 25°

Specifically, the biotin DNA solution 50 μl was injected into the flowcell of the BIACORE-X system in which the sensor chip SA is set, andthen the biotin DNA solution 30 μl was further injected. Thereafter, inorder to flush the biotin DNA non-specifically absorbed to the sensorchip SA, NaOH of 10 mM was injected into the flow cell appropriately,and the sensor chip SA was cleaned.

FIG. 16 is a diagram illustrating a sensorgram obtained in the BIACORE-Xsystem. In the sensorgram, a horizontal axis indicates a time axis, anda vertical axis indicates a mass change. A resonance unit (RU) that is aunit used in the BIACORE-X system was used as a unit of the verticalaxis. 1 RU indicates that there was a mass change of 1 pg per 1 mm². InFIG. 16, for convenience of description, a timing at which the biotinDNA solution 50 μl was putted into, a timing at which the biotin DNAsolution 30 μl was further putted into, and a timing at which NaOH of 50mM was injected are illustrated.

In the sensorgram, an amount of increase in an RU between before thebiotin DNA is injected and after cleaning by NaOH of 50 mM was performedis indicated by an immobilized amount of the biotin DNA. In the exampleillustrated in FIG. 16, ΔRU was about “1270,” and the immobilized amountof the biotin DNA was about 1270 pg/mm².

SPR Measurement

The SPR measuring was performed using the sensor chip SA to which thebiotin DNA is immobilized. Specifically, as shown in Examples 1 to 9 ofTable 1, the analyte solution in which the concentration of the ATP is[y] was prepared by mixing the ATP aptamer complementary strand DNAmixed liquid prepared using the complementary strand DNA[x] with theATP. Then, the prepared analyte solution 35 μl was injected into theflow cell of the BIACORE-X system. The following condition was used whenthe measuring was performed using the BIACORE-X system. The measurementresults are illustrated in FIGS. 19 and 20.

Running buffer: 50 mM Tris (Tris-(hydroxymethyl)aminomethane), 500 mMNaCl, and 5 mM MgCl₂

Velocity: 5 μl/min

Temperature: 25°

TABLE 1 Comparative Example 1 Comparative Example 2 Comparative Example3 Comparative Example 4 Comparative Example 5 Comparative Example 6Comparative Example 7 Comparative Example 8 Example 1 Example 2 Example3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 ATPsolution complementary strand DNA solution ATP aptamer complementarystrand DNA mixed liquid complementary strand DNA[x] complementary strandA complementary strand B complementary strand C complementary strand Acomplementary strand A complementary strand A complementary strand Bcomplementary strand B complementary strand B complementary strand Ccomplementary strand C complementary strand C ATP concentration [y]

Comparative Examples 1 to 5

In Comparative Examples 1 to 5, as will be described below in detail, abiotin ATP aptamer solution was prepared. Thereafter, immobilization ofthe biotin ATP aptamer onto the sensor chip SA and checking of theimmobilization were performed, and SPR measuring was performed.

In other words, in Comparative Examples 1 to 5, unlike Examples 1 to 9,the biotin ATP aptamer was immobilized to the sensor chip SA instead ofthe biotin DNA. As a result, in Examples 1 to 9, the complementarystrand DNA is combined with the biotin DNA immobilized onto the sensorchip SA, whereas in Comparative Examples 1 to 5, the ATP is combinedwith the biotin ATP aptamer immobilized to the sensor chip SA. Further,the biotin ATP aptamer has the base sequence described as the sequencenumber 1 in the sequence listing, and has biotin added to the 5 terminalside of the base sequence. The biotin ATP aptamer was obtained by customsynthesis (Gene Design Inc.).

Preparation of Biotin ATP Aptamer Solution

A biotin ATP aptamer solution 200 μl was prepared by mixing the biotinATP aptamer 1 μl of 10 mM with the HBS-N buffer (BIACORE) 199 μl. In theprepared biotin ATP aptamer solution, the final concentration of thebiotin ATP aptamer was 5 μM.

Immobilization of Biotin ATP Aptamer onto Sensor Chip and Checking ofImmobilization

In the BIACORE-X system, the biotin ATP aptamer was immobilized to thesensor chip SA using strong affinity between streptavidin and biotin.Further, SPR measuring was performed using the BIACORE-X, and theimmobilization of the biotin ATP aptamer was checked based on thesensorgram obtained as a measurement result. The following condition wasused when the measuring was performed using the BIACORE-X system,similarly to the checking of the immobilization of the biotin DNA inExamples 1 to 9.

Running buffer: HBS-N buffer (BIACORE)

Velocity: 5 μl/min

Temperature: 25°

Specifically, the biotin ATP aptamer solution 50 μl was injected intothe flow cell of the BIACORE-X system in which the sensor chip SA isset, and then the biotin ATP aptamer solution 30 μl was furtherinjected. Thereafter, in order to flush the biotin ATP aptamer absorbedto the sensor chip SA regardless of a covalent bond, NaOH of 50 mM wasinjected into the flow cell, and the sensor chip SA was cleaned.

FIG. 17 is a diagram illustrating a sensorgram obtained in the BIACORE-Xsystem. In the sensorgram, a horizontal axis indicates a time axis, anda vertical axis indicates a mass change. An RU that is a unit used inthe BIACORE-X system was used as a unit of the vertical axis. 1 RUindicates that there was a mass change of 1 pg per 1 mm². In FIG. 17,for convenience of description, a timing at which the biotin ATP aptamersolution 50 μl was putted into, a timing at which the biotin ATP aptamersolution 30 μl was putted into, and a timing at which NaOH of 50 mM wasinjected are illustrated.

In the sensorgram, an amount of increase in an RU between before thebiotin ATP aptamer solution is injected and after cleaning by NaOH of 50mM was performed is indicated by an immobilized amount of the biotin ATPaptamer solution. In the example illustrated in FIG. 17, ΔRU was about“350,” and the immobilized amount of the biotin DNA was about 350pg/mm².

ATP Measurement Process

As shown in Comparative Examples 1 to 5 of Table 1, an ATP solution inwhich the concentration of the ATP is [y] was prepared, and the preparedATP solution 35 μl was injected into the flow cell of the BIACORE-Xsystem in which the sensor chip SA to which the biotin ATP aptamer isimmobilized is set. The following condition was used when the measuringwas performed using the BIACORE-X system, similarly to the ATPmeasurement process in Examples 1 to 9. The measurement results areillustrated in FIG. 18.

Running buffer: 50 mM Tris (Tris-(hydroxymethyl)aminomethane), 500 mMNaCl, and 5 mM MgCl₂

Velocity: 5 μl/min

Temperature: 25°

Comparative Example 6 to 8

In Comparative Examples 6 to 8, as will be described below in detail,unlike Examples 1 to 9, a complementary strand DNA solution includingonly a complementary strand DNA[x] of 5 mM was injected at the time ofSPR measuring. It was used as the analyte solution.

In other words, in Examples 1 to 9, the ATP aptamer and thecomplementary strand DNA was annealed, the liquid mixed with the ATP wasprepared, and then the ATP aptamer and the complementary strand DNA wereinjected, whereas in Comparative Examples 6 to 8, only the complementarystrand DNA was injected. Comparative Examples 6 to 8 correspond topositive control of Examples 1 to 9.

Specifically, as shown in Comparative Examples 6 to 8 of Table 1, acomplementary strand DNA solution including a complementary strandDNA[x] of 5 mM was prepared. Then, the prepared complementary strand DNAsolution 35 μl was injected into the flow cell of the BIACORE-X systemin which the sensor chip SA to which the biotin DNA is immobilized isset. The measurement condition used in the BIACORE-X system is asfollows, similarly to the SPR measuring in Examples 1 to 9. Themeasurement results are illustrated in FIGS. 19 and 20.

Running buffer: 50 mM Tris (Tris-(hydroxymethyl)aminomethane), 500 mMNaCl, and 5 mM MgCl₂

Velocity: 5 μl/min

Temperature: 25°

Measurement Results of SPR Measurement

FIG. 18 is a diagram illustrating sensorgrams obtained in ComparativeExamples 1 to 5 in Table 1. In other words, the measurement results whenthe biotin ATP aptamer was immobilized to the sensor chip SA, and thenthe ATP solution was injected are illustrated. As illustrated in FIG.18, a weight change caused by combination of the biotin ATP aptamerimmobilized to the sensor chip SA and the ATP was not measured.

FIG. 19 is a diagram illustrating sensorgrams obtained in Examples 1 to3 and Comparative Example 6 serving as the positive control in Table 1.In other words, the measurement results when the complementary strandDNA “A” is used as the complementary strand DNA[x] are illustrated. Asillustrated in FIG. 19, a weight change caused by a combination of thebiotin DNA immobilized to the sensor chip SA and the complementarystrand DNA disassociated as the ATP is combined with the ATP aptamerwhen the complementary strand DNA “A” is used was measured.

FIG. 20 is a diagram illustrating ΔRU in Examples 1 to 9 and ComparativeExamples 6 to 8 serving as the positive control in Table 1. In otherwords, the measurement results when the complementary strand DNAs “A,”“B,” and “C” are used as the complementary strand DNA[x] areillustrated. As illustrated in FIG. 20, for the complementary strand DNA“A,” a weight change according to a change in a concentration of the ATPwas detected, but for the complementary strand DNAs “B” and “C,” aweight change was not detected.

In other words, as illustrated in FIGS. 18 to 20, it is possible todetect a small molecule having a relatively small molecular weight bydetecting the change of the substrate surface caused by a combinationwith the complementary strand DNA other than the ATP. Further, asillustrated in FIGS. 19 and 20, an amount of an ATP serving as a targetsubstance and a change amount of the detected state change of thesubstrate surface were found to have a proportional relation. As aresult, as illustrated in FIGS. 19 and 20, it is possible to measure anamount of a small molecule serving as a target substance.

REFERENCE SIGNS LIST

1 FIRST COVER MEMBER

2 SECOND COVER MEMBER

3 DETECTING ELEMENT

4 CONCAVE PORTION FORMING THROUGH HOLE

5 CONCAVE PORTION

8 NOTCH

10 SUBSTRATE

11 FIRST IDT ELECTRODE

12 SECOND IDT ELECTRODE

13 DETECTING PORTION

14 INLET

15 GROOVE PORTION

100 SENSOR

210 FIRST SUBSTANCE

220 SECOND SUBSTANCE

230 APTAMER

231 FIRST COMBINING PART

232 SECOND COMBINING PART

240 COMBINING PORTION

300 APTAMER

310 COMBINING PORTION

The invention claimed is:
 1. A sensor, comprising: a substrate, at leastone of an aptamer, a protein and an antibody, a second substance havinga molecular weight larger than a molecular weight of a first substance,and a combining portion disposed on a surface of the substrate, whereinthe combining portion is not initially combined with the secondsubstance, wherein the combining portion is capable of combining withthe second substance, wherein the combining portion is disposed on thesurface of the substrate for detecting whether or not the firstsubstance is included in an analyte having come into contact with bothat least one of an aptamer, a protein, and an antibody and the secondsubstance, wherein the at least one of an aptamer, a protein, and anantibody includes a first combining part for the first substance and asecond combining part for the second substance and is capable of beingcombined with either of the first substance and the second substance,and wherein when the first substance is included in the analyte itcombines to the at least one of an aptamer, a protein, and an antibody,and the second substance combines with the combining portion disposed onthe surface of the substrate.
 2. The sensor according to claim 1,wherein in the aptamer, at least a part of the first combining part andat least a part of the second combining part are a same part.
 3. Thesensor according to claim 1, wherein the at least one of an aptamer, aprotein, and an antibody is preferentially combined with either of thefirst substance and the combining portion.
 4. The sensor according toclaim 1, wherein a first free energy change calculated from adissociation constant of the first substance and the at least one of anaptamer, a protein, and an antibody is smaller than a second free energychange associated with a combination of the at least one of an aptamer,a protein, and an antibody and the second substance, and a third freeenergy change associated with a combination of the second substance andthe combining portion is larger than the second free energy change. 5.The sensor according to claim 4, wherein each of the at least one of anaptamer, a protein, and an antibody and the combining portion has a basesequence, the base sequence of the at least one of an aptamer, aprotein, and an antibody has a part complementary to a first part of abase sequence of the second substance, and the base sequence of thecombining portion has a part complementary to a second part of the basesequence of the second substance, the second free energy change is freeenergy change associated with a combination of the first part of thebase sequence of the second substance and the complementary part of thebase sequence of at least one of an aptamer, a protein, and an antibody,the third free energy change is free energy change associated with acombination of the second part of the base sequence of the secondsubstance and the complementary part of the base sequence of thecombining portion, and a base type and a base number of a base sequencecomplementary between the at least one of an aptamer, a protein, and anantibody and the second substance and a base type and a base number of abase sequence complementary between the combining portion and the secondsubstance have values satisfying a magnitude relation among the firstfree energy change, the second free energy change, and the third freeenergy change.
 6. The sensor according to claim 1, further comprising: afirst cover member in which the substrate is positioned on an uppersurface; and a second cover member bonded to the first cover member,wherein at least one of the first cover member and the second covermember comprises an inlet into which the analyte flows, and a flowchannel extending from the inlet to at least the surface of thesubstrate is formed between the first cover member and the second covermember.
 7. The sensor according to claim 6, wherein the flow channelincludes a groove portion formed on a surface of at least one of thefirst cover member and the second cover member.
 8. The sensor accordingto claim 7, wherein the first cover member comprises a concave portionaccommodating at least a part of the substrate on the upper surface, andthe second cover member comprises the groove portion.
 9. The sensoraccording to claim 7, wherein the at least one of an aptamer, a protein,and an antibody and the second substance are each attached to the grooveportion, and the combining portion is configured to detect the firstsubstance from the analyte that has come into contact with the at leastone of an aptamer, a protein, and an antibody and the second substanceattached to the groove portion.
 10. The sensor according to claim 7,wherein the at least one of an aptamer, a protein, and an antibody isimmobilized to the groove portion, and combined with the secondsubstance, and the combining portion is configured to detect the firstsubstance from the analyte that has come into contact with the at leastone of an aptamer, a protein, and an antibody.
 11. The sensor accordingto claim 10, wherein the at least one of an aptamer, a protein, and anantibody being combined with the second substance is chemically combinedwith a surface substance of the groove portion.
 12. The sensor accordingto claim 1, further comprising: a first InterDigital Transducer (IDT)electrode that is positioned on the surface of the substrate and isconfigured to generate an acoustic wave propagating toward a detectingportion on which the combining portion is positioned on the surface ofthe substrate; and a second IDT electrode that is positioned on thesurface of the substrate, and is configured to receive the acoustic wavehaving passed through the detecting portion.
 13. The sensor according toclaim 12, further comprising: a first bonding member that is bonded toan upper surface of the substrate, and constitutes a first oscillationspace hermetically-sealed between the first bonding member and the uppersurface of the substrate; and a second bonding member that is bonded tothe upper surface of the substrate, and constitutes a second oscillationspace hermetically-sealed between the second bonding member and theupper surface of the substrate, wherein the first oscillation space ispositioned on the first IDT electrode, and the second oscillation spaceis positioned on the second IDT electrode.
 14. The sensor according toclaim 1, wherein at least one of the at least one of an aptamer, aprotein, and an antibody and the analyte is positioned away from thecombining portion.
 15. The sensor according to claim 14, wherein atleast one of the at least one of an aptamer, a protein, and an antibodyand the analyte is positioned in the flow channel.
 16. The sensoraccording to claim 12, wherein at least one of the at least one of anaptamer, a protein, and an antibody and the analyte is positioned in thedetecting portion.